Regulation of human prostasin-like serine protease

ABSTRACT

Reagents which regulate human prostasin-like enzyme activity and reagents which bind to human prostasin-like enzyme gene products can be used to regulate human prostasin-like enzyme activity. Such regulation is particularly useful for treating metastasis of malignant cells, tumor angiogenesis, inflammation, atherosclerosis, neurodegenerative diseases, and pathogenic infections.

This application is a division of Ser. No. 10/311,951 filed Dec. 18,2002. Ser. No. 10/311,951 is a National Stage application of co-pendingPCT application PCT/EP01/07116 filed Jun. 22, 2001, which was publishedin English under PCT Article 21(2) on Dec. 27, 2001 and which claims thebenefit of U.S. provisional application Ser. No. 60/213,474 filed Jun.23, 2000 and Ser. No. 60/277,612 filed Mar. 22, 2001. Each of theseapplications is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the area of enzyme regulation. Moreparticularly, the invention relates to the regulation of humanprostasin-like enzyme activity.

BACKGROUND OF THE INVENTION

Serine proteases are involved in a variety of biological functions,including cell-cell communication, cell migration, and tissueremodeling. Thus, there is a need in the art to identify additionalhuman serine proteases which can be regulated to provide therapeuticeffects.

SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods ofregulating human prostasin-like enzyme activity. These and other objectsof the invention are provided by one or more of the embodimentsdescribed below.

One embodiment of the invention is a prostasin-like enzyme polypeptidecomprising an amino acid sequence selected from the group consisting of:

-   amino acid sequences which are at least about 50% identical to the    amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6; and-   the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6.

Yet another embodiment of the invention is a method of screening foragents which decrease extracellular matrix degradation. A test compoundis contacted with a prostasin-like enzyme polypeptide comprising anamino acid sequence selected from the group consisting of:

-   amino acid sequences which are at least about 50% identical to the    amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6; and-   the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6.

Binding between the test compound and the prostasin-like enzymepolypeptide is detected. A test compound which binds to theprostasin-like enzyme polypeptide is thereby identified as a potentialagent for decreasing extracellular matrix degradation. The agent canwork by decreasing the activity of the prostasin-like enzyme.

Another embodiment of the invention is a method of screening for agentswhich decrease extracellular matrix degradation. A test compound iscontacted with a polynucleotide encoding a prostasin-like enzymepolypeptide, wherein the polynucleotide comprises a nucleotide sequenceselected from the group consisting of:

-   nucleotide sequences which are at least about 50% identical to the    nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; and-   the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5.

Binding of the test compound to the polynucleotide is detected. A testcompound which binds to the polynucleotide is identified as a potentialagent for decreasing extracellular matrix degradation. The agent canwork by decreasing the amount of the prostasin-like enzyme throughinteracting with the prostasin-like enzyme mRNA.

Another embodiment of the invention is a method of screening for agentswhich regulate extracellular matrix degradation. A test compound iscontacted with a prostasin-like enzyme polypeptide comprising an aminoacid sequence selected from the group consisting of:

-   amino acid sequences which are at least about 50% identical to the    amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6; and-   the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6.

A prostasin-like enzyme activity of the polypeptide is detected. A testcompound which increases prostasin-like enzyme activity of thepolypeptide relative to prostasin-like enzyme activity in the absence ofthe test compound is thereby identified as a potential agent forincreasing extracellular matrix degradation. A test compound whichdecreases prostasin-like enzyme activity of the polypeptide relative toprostasin-like enzyme activity in the absence of the test compound isthereby identified as a potential agent for decreasing extracellularmatrix degradation.

Even another embodiment of the invention is a method of screening foragents which decrease extracellular matrix degradation. A test compoundis contacted with a prostasin-like enzyme product of a polynucleotidewhich comprises a nucleotide sequence selected from the group consistingof:

-   nucleotide sequences which are at least about 50% identical to the    nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; and-   the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5.

Binding of the test compound to the prostasin-like enzyme product isdetected. A test compound which binds to the prostasin-like enzymeproduct is thereby identified as a potential agent for decreasingextracellular matrix degradation.

Still another embodiment of the invention is a method of reducingextracellular matrix degradation. A cell is contacted with a reagentwhich specifically binds to a polynucleotide encoding a prostasin-likeenzyme polypeptide or the product encoded by the polynucleotide, whereinthe polynucleotide comprises a nucleotide sequence selected from thegroup consisting of:

-   nucleotide sequences which are at least about 50% identical to the    nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; and-   the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5.

Prostasin-like enzyme activity in the cell is thereby decreased.

The invention thus provides reagents and methods for regulating humanprostasin-like enzyme activity which can be used inter alia, to suppressmetastatic activity of malignant cells, to treat COPD and to enhancehuman prostasin-like enzyme activity during development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA-sequence encoding a prostasin-like enzymepolypeptide (SEQ ID NO:1).

FIG. 2 shows the amino acid sequence deduced from the DNA-sequence ofFIG. 1 (SEQ ID NO:2).

FIG. 3 shows the amino acid sequence of the protein identified by SwissProt Accession No. Q16651 (SEQ ID NO:3).

FIG. 4 shows the DNA-sequence encoding a prostasin-like enzymepolypeptide (SEQ ID NO: 4).

FIG. 5 shows the DNA-sequence encoding a prostasin-like enzymepolypeptide (SEQ ID NO: 5).

FIG. 6 shows the amino acid sequence deduced from the DNA-sequence ofFIG. 5 (SEQ ID NO: 6).

FIG. 7 shows the alignment of prostasin-like enzyme as shown in SEQ IDNO:2 with the human protein identified by Swiss Prot Accession No.Q16651 (SEQ ID NO:3).

FIG. 8 shows the prosite search results.

FIG. 9 shows the BLOCKS search results for SEQ ID NO:2.

FIG. 10 shows the BLAST alignment of SEQ ID NO:2 againstswiss/Q16651/PSS8_HUMAN SEQ ID NO:3).

FIG. 11 shows the gene prediction for SEQ ID NO:1 based on EST BF689477(SEQ ID NO:5)

FIG. 12 shows the relative expression of prostasin-like enzyme invarious human tissues.

FIG. 13 shows the relative expression of prostasin-like enzyme invarious human respiratory tissues and cells. Key: HBEC=cultured humanbronchial epithelial cells; H441=Clara-like cells, SMC=cultured airwaysmooth muscle cells; SAE=cultured small airway epithelial cells;AII=primary cultured alveolar type II cells; PMN=polymorphonuclearleukocytes; Mono=monocytes; Cult Mono=cultured monocytes(macrophage-like).

DETAILED DESCRIPTION OF INVENTION

The invention relates to an isolated polynucleotide encoding aprostasin-like enzyme polypeptide and being selected from the groupconsisting of:

-   1. a polynucleotide encoding a prostasin-like enzyme polypeptide    comprising an amino acid sequence selected from the group consisting    of:    -   amino acid sequences which are at least about 50% identical to    -   the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6;        and    -   the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6.-   2. a polynucleotide comprising the sequence of SEQ ID NO: 1 or SEQ    ID NO: 5;-   3. a polynucleotide which hybridizes under stringent conditions to a    polynucleotide specified in (a) and (b);-   4. a polynucleotide the sequence of which deviates from the    polynucleotide sequences specified in (a) to (c) due to the    degeneration of the genetic code; and-   5. a polynucleotide which represents a fragment, derivative or    allelic variation of a polynucleotide sequence specified in (a) to    (d).

Furthermore, it has been discovered by the present applicant that anovel prostasin-like enzyme, particularly a human prostasin-like enzyme,is a discovery of the present invention. Human prostasin-like enzyme asshown in SEQ ID NO:2 is 47% identical over 243 amino acids to the humanprotein identified by SwissProt Accession No. Q16651 (SEQ ID NO:3) andannotated as a prostasin precursor (FIG. 1). Prostasin is a trypsin-likeserine protease which has been purified from human seminal fluid and islocalized in epithelial cells and ducts of the prostate gland, as wellas in colon, lung, kidney, pancreas, salivary gland, liver, and bronchi.(Yu et al., J. Biol. Chem. 269, 11843-48, 1994). Human prostasin-likeenzyme contains a trypsin-serine and a trypsin-histidine region as shownin FIG. 1. Other domains identified in human prostasin-like enzymeinclude fibronectin domains, Kringle domains, and Apple domains, asshown in FIG. 3. Thus, human prostasin-like enzyme is believed to beuseful for the same purposes as previously identified serine proteases,including prostasin. Regulation of human prostasin-like enzyme can beused, for example, to treat conditions such as osteoporosis, COPD, tumormetastasis, and neurodegenerative diseases.

Polypeptides

Human prostasin-like enzyme polypeptides according to the inventioncomprise at least 6, 10, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225,250, or 260 contiguous amino acids selected from the amino acid sequenceshown in SEQ ID NO:2 or SEQ ID NO: 6 or a biologically active variantthereof, as defined below. A human prostasin-like enzyme polypeptide ofthe invention therefore can be a portion of a human prostasin-likeenzyme protein, a full-length human prostasin-like enzyme protein, or afusion protein comprising all or a portion of a human prostasin-likeenzyme protein.

Biologically Active Variants

Human prostasin-like enzyme polypeptide variants which are biologicallyactive, e.g., retain a serine protease activity, also are humanprostasin-like enzyme polypeptides. Preferably, naturally ornon-naturally occurring prostasin-like enzyme polypeptide variants haveamino acid sequences which are at least about 50, 55, 60, 65, or 70,preferably about 75, 80, 85, 90, 96, 96, or 98% identical to the aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO: 6 or a fragmentthereof. Percent identity between a putative human prostasin-like enzymepolypeptide variant and an amino acid sequence of SEQ ID NO:2 or SEQ IDNO: 6 is determined using the Blast2 alignment program (Blosum62, Expect10, standard genetic codes).

Variations in percent identity can be due, for example, to amino acidsubstitutions, insertions, or deletions. Amino acid substitutions aredefined as one for one amino acid replacements. They are conservative innature when the substituted amino acid has similar structural and/orchemical properties. Examples of conservative replacements aresubstitution of a leucine with an isoleucine or valine, an aspartatewith a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an aminoacid sequence. They typically fall in the range of about 1 to 5 aminoacids. Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without abolishing biological orimmunological activity of a human prostasin-like enzyme polypeptide canbe found using computer programs well known in the art, such as DNASTARsoftware. Whether an amino acid change results in a biologically activehuman prostasin-like enzyme polypeptide can readily be determined byassaying for serine protease activity, as described for example, in thespecific Examples, below.

Fusion Proteins

Fusion proteins are useful for generating antibodies against humanprostasin-like enzyme polypeptide amino acid sequences and for use invarious assay systems. For example, fusion proteins can be used toidentify proteins which interact with portions of a human prostasin-likeenzyme polypeptide. Protein affinity chromatography or library-basedassays for protein-protein interactions, such as the yeast two-hybrid orphage display systems, can be used for this purpose. Such methods arewell known in the art and also can be used as drug screens.

A human prostasin-like enzyme polypeptide fusion protein comprises twopolypeptide segments fused together by means of a peptide bond. Thefirst polypeptide segment comprises at least 6, 10, 15, 25, 50, 75, 100,125, 150, 175, 200, 225, 250, or 260 contiguous amino acids of SEQ IDNO:2 or SEQ ID NO: 6 or of a biologically active variant, such as thosedescribed above. The first polypeptide segment also can comprisefull-length human prostasin-like human prostasin-like enzyme protein.

The second polypeptide segment can be a full-length protein or a proteinfragment. Proteins commonly used in fusion protein construction includeβ-galactosidase, β-glucuronidase, green fluorescent protein (GFP),autofluorescent proteins, including blue fluorescent protein (BFP),glutathione-S-transferase (GST), luciferase, horse-radish peroxidase(HRP), and chloramphenicol acetyltransferase (CAT). Additionally,epitope tags are used in fusion protein constructions, includinghistidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myctags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructionscan include maltose binding protein (MBP), S-tag, Lex a DNA bindingdomain (DBD) fusions, GAL4 DNA binding domain fusions, and herpessimplex virus (HSV) BP16 protein fusions. A fusion protein also can beengineered to contain a cleavage site located between the humanprostasin-like enzyme polypeptide-encoding sequence and the heterologousprotein sequence, so that the human prostasin-like enzyme polypeptidecan be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art.Preferably, a fusion protein is produced by covalently linking twopolypeptide segments or by standard procedures in the art of molecularbiology. Recombinant DNA methods can be used to prepare fusion proteins,for example, by making a DNA construct which comprises coding sequencesselected from SEQ ID NO:1 or SEQ ID NO: 5 in proper reading frame withnucleotides encoding the second polypeptide segment and expressing theDNA construct in a host cell, as is known in the art. Many kits forconstructing fusion proteins are available from companies such asPromega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.),CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz,Calif.), MBL International Corporation (MIC; Watertown, Mass.), andQuantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Identification of Species Homologs

Species homologs of human prostasin-like enzyme polypeptide can beobtained using human prostasin-like enzyme polypeptide polynucleotides(described below) to make suitable probes or primers for screening cDNAexpression libraries from other species, such as mice, monkeys, oryeast, identifying cDNAs which encode homologs of human prostasin-likeenzyme polypeptide, and expressing the cDNAs as is known in the art.

Polynucleotides

A human prostasin-like enzyme polynucleotide can be single- ordouble-stranded and comprises a coding sequence or the complement of acoding sequence for a human prostasin-like enzyme polypeptide. Afull-length coding sequence for human prostasin-like enzyme is shown inSEQ ID NO:1 and SEQ ID NO: 5.

Degenerate nucleotide sequences encoding human prostasin-like enzymepolypeptides, as well as homologous nucleotide sequences which are atleast about 50, preferably about 75, 90, 96, or 98% identical to thenucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO: 5 also are humanprostasin-like enzyme polynucleotides. Percent sequence identity betweenthe sequences of two polynucleotides is determined using computerprograms such as ALIGN which employ the FASTA algorithm, using an affinegap search with a gap open penalty of −12 and a gap extension penalty of−2. Complementary DNA (cDNA) molecules, species homologs, and variantsof human prostasin-like enzyme polypeptides also are humanprostasin-like enzyme polynucleotides.

Identification of Polynucleotide Variants and Homologs

Variants and homologs of the human prostasin-like enzyme polynucleotidesdescribed above also are human prostasin-like enzyme polynucleotides.Typically, homologous human prostasin-like enzyme polynucleotidesequences can be identified by hybridization of candidatepolynucleotides to known human prostasin-like enzyme polynucleotidesunder stringent conditions, as is known in the art. For example, usingthe following wash conditions—2×SSC (0.3 M NaCl, 0.03 M sodium citrate,pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2×SSC,0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, room temperature twice,10 minutes each—homologous sequences can be identified which contain atmost about 25-30% basepair mismatches. More preferably, homologousnucleic acid strands contain 15-25% basepair mismatches, even morepreferably 5-15% basepair mismatches.

Species homologs of the human prostasin-like enzyme polynucleotidesdisclosed herein also can be identified by making suitable probes orprimers and screening cDNA expression libraries from other species, suchas mice, monkeys, or yeast. Human variants of human prostasin-likeenzyme polynucleotides can be identified, for example, by screeninghuman cDNA expression libraries. It is well known that the T_(m) of adouble-stranded DNA decreases by 1-1.5° C. with every 1% decrease inhomology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Variants of humanprostasin-like enzyme polynucleotides or human prostasin-like enzymepolynucleotides of other species can therefore be identified byhybridizing a putative homologous human prostasin-like enzymepolynucleotide with a polynucleotide having a nucleotide sequence of SEQID NO:1 or SEQ ID NO: 5 or the complement thereof to form a test hybrid.The melting temperature of the test hybrid is compared with the meltingtemperature of a hybrid comprising polynucleotides having perfectlycomplementary nucleotide sequences, and the number or percent ofbasepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to human prostasin-like enzymepolynucleotides or their complements following stringent hybridizationand/or wash conditions also are human prostasin-like enzymepolynucleotides. Stringent wash conditions are well known and understoodin the art and are disclosed, for example, in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination oftemperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m) of the hybrid understudy. The T_(m) of a hybrid between a human prostasin-like enzymepolynucleotide having a nucleotide sequence shown in SEQ ID NO:1 or SEQID NO: 5 or the complement thereof and a polynucleotide sequence whichis at least about 50, preferably about 75, 90, 96, or 98% identical toone of those nucleotide sequences can be calculated, for example, usingthe equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48,1390 (1962):T _(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−0.63(% formamide)−600/l),where l=the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4×SSC at 65° C., or 50%formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highlystringent wash conditions include, for example, 0.2×SSC at 65° C.

Preparation of Polynucleotides

A naturally occurring human prostasin-like enzyme polynucleotide can beisolated free of other cellular components such as membrane components,proteins, and lipids. Polynucleotides can be made by a cell and isolatedusing standard nucleic acid purification techniques, or synthesizedusing an amplification technique, such as the polymerase chain reaction(PCR), or by using an automatic synthesizer. Methods for isolatingpolynucleotides are routine and are known in the art. Any such techniquefor obtaining a polynucleotide can be used to obtain isolated humanprostasin-like enzyme polynucleotides. For example, restriction enzymesand probes can be used to isolate polynucleotide fragments whichcomprises human prostasin-like enzyme nucleotide sequences. Isolatedpolynucleotides are in preparations which are free or at least 70, 80,or 90% free of other molecules.

Human prostasin-like enzyme cDNA molecules can be made with standardmolecular biology techniques, using human prostasin-like enzyme mRNA asa template. human prostasin-like enzyme cDNA molecules can thereafter bereplicated using molecular biology techniques known in the art anddisclosed in manuals such as Sambrook et al. (1989). An amplificationtechnique, such as PCR, can be used to obtain additional copies ofpolynucleotides of the invention, using either human genomic DNA or cDNAas a template.

Alternatively, synthetic chemistry techniques can be used to synthesizeshuman prostasin-like enzyme polynucleotides. The degeneracy of thegenetic code allows alternate nucleotide sequences to be synthesizedwhich will encode a human prostasin-like enzyme polypeptide having, forexample, an amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO: 6 ora biologically active variant thereof.

Extending Polynucleotides

Various PCR-based methods can be used to extend the nucleic acidsequences disclosed herein to detect upstream sequences such aspromoters and regulatory elements. For example, restriction-site PCRuses universal primers to retrieve unknown sequence adjacent to a knownlocus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA isfirst amplified in the presence of a primer to a linker sequence and aprimer specific to the known region. The amplified sequences are thensubjected to a second round of PCR with the same linker primer andanother specific primer internal to the first one. Products of eachround of PCR are transcribed with an appropriate RNA polymerase andsequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia et al., Nucleic AcidsRes. 16, 8186, 1988). Primers can be designed using commerciallyavailable software, such as OLIGO 4.06 Primer Analysis software(National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which can be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic.1, 111-119, 1991). In this method, multiple restriction enzymedigestions and ligations also can be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which can be used to retrieve unknown sequences is thatof Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991). Additionally,PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto,Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.).This process avoids the need to screen libraries and is useful infinding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Randomly-primedlibraries are preferable, in that they will contain more sequences whichcontain the 5′ regions of genes. Use of a randomly primed library may beespecially preferable for situations in which an oligo d(T) library doesnot yield a full-length cDNA. Genomic libraries can be useful forextension of sequence into 5′ non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used toanalyze the size or confirm the nucleotide sequence of PCR or sequencingproducts. For example, capillary sequencing can employ flowable polymersfor electrophoretic separation, four different fluorescent dyes (one foreach nucleotide) which are laser activated, and detection of the emittedwavelengths by a charge coupled device camera. Output/light intensitycan be converted to electrical signal using appropriate software (e.g.GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire processfrom loading of samples to computer analysis and electronic data displaycan be computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA which might bepresent in limited amounts in a particular sample.

Obtaining Polypeptides

Human prostasin-like enzyme polypeptides can be obtained, for example,by purification from human cells, by expression of human prostasin-likeenzyme polynucleotides, or by direct chemical synthesis.

Protein Purification

Human prostasin-like enzyme polypeptides can be purified from any cellwhich expresses the enzyme, including host cells which have beentransfected with human prostasin-like enzyme expression constructs.Human fetal heart provides an especially useful source of humanprostasin-like enzyme polypeptides. A purified human prostasin-likeenzyme polypeptide is separated from other compounds which normallyassociate with the human prostasin-like enzyme polypeptide in the cell,such as certain proteins, carbohydrates, or lipids, using methodswell-known in the art. Such methods include, but are not limited to,size exclusion chromatography, ammonium sulfate fractionation, ionexchange chromatography, affinity chromatography, and preparative gelelectrophoresis. A preparation of purified human prostasin-like enzymepolypeptides is at least 80% pure; preferably, the preparations are 90%,95%, or 99% pure. Purity of the preparations can be assessed by anymeans known in the art, such as SDS-polyacrylamide gel electrophoresis.

Expression of Polynucleotides

To express a human prostasin-like enzyme polynucleotide, thepolynucleotide can be inserted into an expression vector which containsthe necessary elements for the transcription and translation of theinserted coding sequence. Methods which are well known to those skilledin the art can be used to construct expression vectors containingsequences encoding human prostasin-like enzyme polypeptides andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed, for example, in Sambrook et al. (1989) and in Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,N.Y., 1989.

A variety of expression vector/host systems can be utilized to containand express sequences encoding a human prostasin-like enzymepolypeptide. These include, but are not limited to, microorganisms, suchas bacteria transformed with recombinant bacteriophage, plasmid, orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors, insect cell systems infected with virus expression vectors(e.g., baculovirus), plant cell systems transformed with virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translatedregions of the vector—enhancers, promoters, 5′ and 3′ untranslatedregions—which interact with host cellular proteins to carry outtranscription and translation. Such elements can vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, can be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.)or pSPORT1 plasmid (Life Technologies) and the like can be used. Thebaculovirus polyhedrin promoter can be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (e.g., heat shock,RUBISCO, and storage protein genes) or from plant viruses (e.g., viralpromoters or leader sequences) can be cloned into the vector. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferable. If it is necessary to generate a cell line thatcontains multiple copies of a nucleotide sequence encoding a humanprostasin-like enzyme polypeptide, vectors based on SV40 or EBV can beused with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selecteddepending upon the use intended for the human prostasin-like enzymepolypeptide. For example, when a large quantity of a humanprostasin-like enzyme polypeptide is needed for the induction ofantibodies, vectors which direct high level expression of fusionproteins that are readily purified can be used. Such vectors include,but are not limited to, multifunctional E. coli cloning and expressionvectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, asequence encoding the human prostasin-like enzyme polypeptide can beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced. pIN vectors (Van Heeke & Schuster, J. Biol. Chem.264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also canbe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems can be designed to includeheparin, thrombin, or factor Xa protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) andGrant et al., Methods Enzymol. 153, 516-544, 1987.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequencesencoding human prostasin-like enzyme polypeptides can be driven by anyof a number of promoters. For example, viral promoters such as the 35Sand 19S promoters of CaMV can be used alone or in combination with theomega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters can be used (Coruzzi et al., EMBO J. 3, 1671-1680,1984; Broglie et al., Science 224, 838-843, 1984; Winter et al., ResultsProbl. Cell Differ. 17, 85-105, 1991). These constructs can beintroduced into plant cells by direct DNA transformation or bypathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (e.g., Hobbs or Murray, in MCGRAWHILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y.,pp. 191-196, 1992).

An insect system also can be used to express a human prostasin-likeenzyme polypeptide. For example, in one such system Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. Sequences encoding human prostasin-like enzyme polypeptides canbe cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of human prostasin-like enzyme polypeptides willrender the polyhedrin gene inactive and produce recombinant viruslacking coat protein. The recombinant viruses can then be used to infectS. frugiperda cells or Trichoplusia larvae in which human prostasin-likeenzyme polypeptides can be expressed (Engelhard et al., Proc. Nat. Acad.Sci. 91, 3224-3227, 1994).

Mammalian Expression Systems

A number of viral-based expression systems can be used to express humanprostasin-like enzyme polypeptides in mammalian host cells. For example,if an adenovirus is used as an expression vector, sequences encodinghuman prostasin-like enzyme polypeptides can be ligated into anadenovirus transcription/translation complex comprising the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome can be used to obtain a viable viruswhich is capable of expressing a human prostasin-like enzyme polypeptidein infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81,3655-3659, 1984). If desired, transcription enhancers, such as the Roussarcoma virus (RSV) enhancer, can be used to increase expression inmammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver largerfragments of DNA than can be contained and expressed in a plasmid. HACsof 6M to 10M are constructed and delivered to cells via conventionaldelivery methods (e.g., liposomes, polycationic amino polymers, orvesicles).

Specific initiation signals also can be used to achieve more efficienttranslation of sequences encoding human prostasin-like enzymepolypeptides. Such signals include the ATG initiation codon and adjacentsequences. In cases where sequences encoding a human prostasin-likeenzyme polypeptide, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals (including the ATG initiationcodon) should be provided. The initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons can be of various origins,both natural and synthetic. The efficiency of expression can be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used (see Scharf et al., Results Probl. CellDiffer. 20, 125-162, 1994).

Host Cells

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed humanprostasin-like enzyme polypeptide in the desired fashion. Suchmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the polypeptide also can be used to facilitate correctinsertion, folding and/or function. Different host cells which havespecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38),are available from the American Type Culture Collection (ATCC; 10801University Boulevard, Manassas, Va. 20110-2209) and can be chosen toensure the correct modification and processing of the foreign protein.

Stable expression is preferred for long-term, high-yield production ofrecombinant proteins. For example, cell lines which stably express humanprostasin-like enzyme polypeptides can be transformed using expressionvectors which can contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells can beallowed to grow for 1-2 days in an enriched medium before they areswitched to a selective medium. The purpose of the selectable marker isto confer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced humanprostasin-like enzyme sequences. Resistant clones of stably transformedcells can be proliferated using tissue culture techniques appropriate tothe cell type. See, for example, ANIMAL CELL CULTURE, R. I. Freshney,ed., 1986.

Any number of selection systems can be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11, 223-32, 1977) and adeninephosphoribosyltransferase (Lowy et al., Cell 22, 817-23, 1980) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77, 3567-70, 1980),npt confers resistance to the aminoglycosides, neomycin and G-418(Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981), and als and patconfer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murray, 1992, supra). Additionalselectable genes have been described. For example, trpB allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, can be used to identify transformants and to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Methods Mol. Biol. 55, 121-131,1995).

Detecting Expression

Although the presence of marker gene expression suggests that the humanprostasin-like enzyme polynucleotide is also present, its presence andexpression may need to be confirmed. For example, if a sequence encodinga human prostasin-like enzyme polypeptide is inserted within a markergene sequence, transformed cells containing sequences which encode ahuman prostasin-like enzyme polypeptide can be identified by the absenceof marker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding a human prostasin-like enzymepolypeptide under the control of a single promoter. Expression of themarker gene in response to induction or selection usually indicatesexpression of the human prostasin-like enzyme polynucleotide.

Alternatively, host cells which contain a human prostasin-like enzymepolynucleotide and which express a human prostasin-like enzymepolypeptide can be identified by a variety of procedures known to thoseof skill in the art. These procedures include, but are not limited to,DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassaytechniques which include membrane, solution, or chip-based technologiesfor the detection and/or quantification of nucleic acid or protein. Forexample, the presence of a polynucleotide sequence encoding a humanprostasin-like enzyme polypeptide can be detected by DNA-DNA or DNA-RNAhybridization or amplification using probes or fragments or fragments ofpolynucleotides encoding a human prostasin-like enzyme polypeptide.Nucleic acid amplification-based assays involve the use ofoligonucleotides selected from sequences encoding a human prostasin-likeenzyme polypeptide to detect transformants which contain a humanprostasin-like enzyme polynucleotide.

A variety of protocols for detecting and measuring the expression of ahuman prostasin-like enzyme polypeptide, using either polyclonal ormonoclonal antibodies specific for the polypeptide, are known in theart. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay using monoclonal antibodiesreactive to two non-interfering epitopes on a human prostasin-likeenzyme polypeptide can be used, or a competitive binding assay can beemployed. These and other assays are described in Hampton et al.,SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn.,1990) and Maddox et al., J. Exp. Med. 158, 1211-1216, 1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding humanprostasin-like enzyme polypeptides include oligolabeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, sequences encoding a human prostasin-likeenzyme polypeptide can be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and can be used to synthesize RNA probes in vitro by additionof labeled nucleotides and an appropriate RNA polymerase such as T7, T3,or SP6. These procedures can be conducted using a variety ofcommercially available kits (Amersham Pharmacia Biotech, Promega, and USBiochemical). Suitable reporter molecules or labels which can be usedfor ease of detection include radionuclides, enzymes, and fluorescent,chemiluminescent, or chromogenic agents, as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of Polypeptides

Host cells transformed with nucleotide sequences encoding a humanprostasin-like enzyme polypeptide can be cultured under conditionssuitable for the expression and recovery of the protein from cellculture. The polypeptide produced by a transformed cell can be secretedor contained intracellularly depending on the sequence and/or the vectorused. As will be understood by those of skill in the art, expressionvectors containing polynucleotides which encode human prostasin-likeenzyme polypeptides can be designed to contain signal sequences whichdirect secretion of soluble human prostasin-like enzyme polypeptidesthrough a prokaryotic or eukaryotic cell membrane or which direct themembrane insertion of membrane-bound human prostasin-like enzymepolypeptide.

As discussed above, other constructions can be used to join a sequenceencoding a human prostasin-like enzyme polypeptide to a nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). Inclusion ofcleavable linker sequences such as those specific for Factor Xa orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and the human prostasin-like enzyme polypeptide also can be usedto facilitate purification. One such expression vector provides forexpression of a fusion protein containing a human prostasin-like enzymepolypeptide and 6 histidine residues preceding a thioredoxin or anenterokinase cleavage site. The histidine residues facilitatepurification by IMAC (immobilized metal ion affinity chromatography, asdescribed in Porath et al., Prot. Exp. Purif. 3, 263-281, 1992), whilethe enterokinase cleavage site provides a means for purifying the humanprostasin-like enzyme polypeptide from the fusion protein. Vectors whichcontain fusion proteins are disclosed in Kroll et al., DNA Cell Biol.12, 441-453, 1993.

Chemical Synthesis

Sequences encoding a human prostasin-like enzyme polypeptide can besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers et al., Nucl. Acids Res. Symp. Ser. 215-223,1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).Alternatively, a human prostasin-like enzyme polypeptide itself can beproduced using chemical methods to synthesize its amino acid sequence,such as by direct peptide synthesis using solid-phase techniques(Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al.,Science 269, 202-204, 1995). Protein synthesis can be performed usingmanual techniques or by automation. Automated synthesis can be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Optionally, fragments of human prostasin-like enzymepolypeptides can be separately synthesized and combined using chemicalmethods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton,PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., NewYork, N.Y., 1983). The composition of a synthetic human prostasin-likeenzyme polypeptide can be confirmed by amino acid analysis or sequencing(e.g., the Edman degradation procedure; see Creighton, supra).Additionally, any portion of the amino acid sequence of the humanprostasin-like enzyme polypeptide can be altered during direct synthesisand/or combined using chemical methods with sequences from otherproteins to produce a variant polypeptide or a fusion protein.

Production of Altered Polypeptides

As will be understood by those of skill in the art, it may beadvantageous to produce human prostasin-like enzyme polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producean RNA transcript having desirable properties, such as a half-life whichis longer than that of a transcript generated from the naturallyoccurring sequence.

The nucleotide sequences disclosed herein can be engineered usingmethods generally known in the art to alter human prostasin-like enzymepolypeptide-encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the polypeptide or mRNA product. DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides can be used to engineer the nucleotide sequences. Forexample, site-directed mutagenesis can be used to insert new restrictionsites, alter glycosylation patterns, change codon preference, producesplice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bindspecifically to an epitope of a human prostasin-like enzyme polypeptide.“Antibody” as used herein includes intact immunoglobulin molecules, aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding an epitope of a human prostasin-like enzymepolypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acidsare required to form an epitope. However, epitopes which involvenon-contiguous amino acids may require more, e.g., at least 15, 25, or50 amino acids.

An antibody which specifically binds to an epitope of a humanprostasin-like enzyme polypeptide can be used therapeutically, as wellas in immunochemical assays, such as Western blots, ELISAs,radioimmunoassays, immunohistochemical assays, inmunoprecipitations, orother immunochemical assays known in the art. Various immunoassays canbe used to identify antibodies having the desired specificity. Numerousprotocols for competitive binding or immunoradiometric assays are wellknown in the art. Such immunoassays typically involve the measurement ofcomplex formation between an immunogen and an antibody whichspecifically binds to the immunogen.

Typically, an antibody which specifically binds to a humanprostasin-like enzyme polypeptide provides a detection signal at least5-, 10-, or 20-fold higher than a detection signal provided with otherproteins when used in an immunochemical assay. Preferably, antibodieswhich specifically bind to human prostasin-like enzyme polypeptides donot detect other proteins in immunochemical assays and canimmunoprecipitate a human prostasin-like enzyme polypeptide fromsolution.

Human prostasin-like enzyme polypeptides can be used to immunize amammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, toproduce polyclonal antibodies. If desired, a human prostasin-like enzymepolypeptide can be conjugated to a carrier protein, such as bovine serumalbumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on thehost species, various adjuvants can be used to increase theimmunological response. Such adjuvants include, but are not limited to,Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surfaceactive substances (e.g. lysolecithin, pluronic polyols, polyanions,peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially useful.

Monoclonal antibodies which specifically bind to a human prostasin-likeenzyme polypeptide can be prepared using any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These techniques include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler et al., Nature 256, 495-497, 1985; Kozbor et al., J.Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci.80, 2026-2030, 1983; Cole et al., Mol. Cell Biol. 62, 109-120, 1984).

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (Morrison et al., Proc. Natl. Acad.Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984;Takeda et al., Nature 314, 452-454, 1985). Monoclonal and otherantibodies also can be “humanized” to prevent a patient from mounting animmune response against the antibody when it is used therapeutically.Such antibodies may be sufficiently similar in sequence to humanantibodies to be used directly in therapy or may require alteration of afew key residues. Sequence differences between rodent antibodies andhuman sequences can be minimized by replacing residues which differ fromthose in the human sequences by site directed mutagenesis of individualresidues or by grating of entire complementarity determining regions.Alternatively, humanized antibodies can be produced using recombinantmethods, as described in GB2188638B. Antibodies which specifically bindto a human prostasin-like enzyme polypeptide can contain antigen bindingsites which are either partially or fully humanized, as disclosed inU.S. Pat. No. 5,565,332.

Alternatively, techniques described for the production of single chainantibodies can be adapted using methods known in the art to producesingle chain antibodies which specifically bind to human prostasin-likeenzyme polypeptides. Antibodies with related specificity, but ofdistinct idiotypic composition, can be generated by chain shuffling fromrandom combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad.Sci. 88, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a templatechirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chainantibodies can be mono- or bispecific, and can be bivalent ortetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught, for example, in Coloma & Morrison, 1997, Nat.Biotechnol. 15, 159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught in Mallender & Voss, 1994, J. Biol.Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology (Verhaar et al., 1995,Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth.165, 81-91).

Antibodies which specifically bind to human prostasin-like enzymepolypeptides also can be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter etal., Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used therapeutically inmethods of the invention. For example, chimeric antibodies can beconstructed as disclosed in WO 93/03151. Binding proteins which arederived from immunoglobulins and which are multivalent andmultispecific, such as the “diabodies” described in WO 94/13804, alsocan be prepared.

Antibodies according to the invention can be purified by methods wellknown in the art. For example, antibodies can be affinity purified bypassage over a column to which a human prostasin-like enzyme polypeptideis bound. The bound antibodies can then be eluted from the column usinga buffer with a high salt concentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences which arecomplementary to a specific DNA or RNA sequence. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form complexes and block either transcription ortranslation. Preferably, an antisense oligonucleotide is at least 11nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40,45, or 50 or more nucleotides long. Longer sequences also can be used.Antisense oligonucleotide molecules can be provided in a DNA constructand introduced into a cell as described above to decrease the level ofhuman prostasin-like enzyme gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides,or a combination of both. Oligonucleotides can be synthesized manuallyor by an automated synthesizer, by covalently lining the 5′ end of onenucleotide with the 3′ end of another nucleotide with non-phosphodiesterinternucleotide linkages such alkylphosphonates, phosphorothioates,phosphorodithioates, alkylphosphonothioates, alkylphosphonates,phosphoramidates, phosphate esters, carbamates, acetamidate,carboxymethyl esters, carbonates, and phosphate triesters. See Brown,Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72,1994; Uhlmann et al., Chem. Rev. 90, 543-583, 1990.

Modifications of human prostasin-like enzyme gene expression can beobtained by designing antisense oligonucleotides which will formduplexes to the control, 5′, or regulatory regions of the humanprostastin-like enzyme gene. Oligonucleotides derived from thetranscription initiation site, e.g., between positions −10 and +10 fromthe start site, are preferred. Similarly, inhibition can be achievedusing “triple helix” base-pairing methodology. Triple helix pairing isuseful because it causes inhibition of the ability of the double helixto open sufficiently for the binding of polymerases, transcriptionfactors, or chaperons. Therapeutic advances using triplex DNA have beendescribed in the literature (e.g., Gee et al., in Huber & Carr,MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco,N.Y., 1994). An antisense oligonucleotide also can be designed to blocktranslation of mRNA by preventing the transcript from binding toribosomes.

Precise complementarity is not required for successful complex formationbetween an antisense oligonucleotide and the complementary sequence of ahuman prostasin-like enzyme polynucleotide. Antisense oligonucleotideswhich comprise, for example, 2, 3, 4, or 5 or more stretches ofcontiguous nucleotides which are precisely complementary to a humanprostasin-like enzyme polynucleotide, each separated by a stretch ofcontiguous nucleotides which are not complementary to adjacent humanprostasin-like enzyme nucleotides, can provide sufficient targetingspecificity for human prostasin-like enzyme mRNA. Preferably, eachstretch of complementary contiguous nucleotides is at least 4, 5, 6, 7,or 8 or more nucleotides in length. Non-complementary interveningsequences are preferably 1, 2, 3, or 4 nucleotides in length. Oneskilled in the art can easily use the calculated melting point of anantisense-sense pair to determine the degree of mismatching which willbe tolerated between a particular antisense oligonucleotide and aparticular human prostasin-like enzyme polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting theirability to hybridize to a human prostasin-like enzyme polynucleotide.These modifications can be internal or at one or both ends of theantisense molecule. For example, internucleoside phosphate linkages canbe modified by adding cholesteryl or diamine moieties with varyingnumbers of carbon residues between the amino groups and terminal ribose.Modified bases and/or sugars, such as arabinose instead of ribose, or a3′, 5′-substituted oligonucleotide in which the 3′ hydroxyl group or the5′ phosphate group are substituted, also can be employed in a modifiedantisense oligonucleotide. These modified oligonucleotides can beprepared by methods well known in the art. See, e.g., Agrawal et al.,Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al., Chem. Rev. 90,543-584, 1990; Uhlmann et al., Tetrahedron Lett. 215, 3539-3542, 1987.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech,Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568;1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture &Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used toinhibit gene function by cleaving an RNA sequence, as is known in theart (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The mechanism ofribozyme action involves sequence-specific hybridization of the ribozymemolecule to complementary target RNA, followed by endonucleolyticcleavage. Examples include engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of specific nucleotide sequences.

The coding sequence of a human prostasin-like enzyme polynucleotide canbe used to generate ribozymes which will specifically bind to mRNAtranscribed from the human prostasin-like enzyme polynucleotide. Methodsof designing and constructing ribozymes which can cleave other RNAmolecules in trans in a highly sequence specific manner have beendeveloped and described in the art (see Haseloff et al. Nature 334,585-591, 1988). For example, the cleavage activity of ribozymes can betargeted to specific RNAs by engineering a discrete “hybridization”region into the ribozyme. The hybridization region contains a sequencecomplementary to the target RNA and thus specifically hybridizes withthe target (see, for example, Gerlach et al., EP 321,201).

Specific ribozyme cleavage sites within a human prostasin-like humanprostasin-like enzyme RNA target can be identified by scanning thetarget molecule for ribozyme cleavage sites which include the followingsequences: GUA, GUU, and GUC. Once identified, short RNA sequences ofbetween 15 and 20 ribonucleotides corresponding to the region of thetarget RNA containing the cleavage site can be evaluated for secondarystructural features which may render the target inoperable. Suitabilityof candidate human prostasin-like enzyme RNA targets also can beevaluated by testing accessibility to hybridization with complementaryoligonucleotides using ribonuclease protection assays. Longercomplementary sequences can be used to increase the affinity of thehybridization sequence for the target. The hybridizing and cleavageregions of the ribozyme can be integrally related such that uponhybridizing to the target RNA through the complementary regions, thecatalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct.Mechanical methods, such as microinjection, liposome-mediatedtransfection, electroporation, or calcium phosphate precipitation, canbe used to introduce a ribozyme-containing DNA construct into cells inwhich it is desired to decrease human prostasin-like enzyme expression.Alternatively, if it is desired that the cells stably retain the DNAconstruct, the construct can be supplied on a plasmid and maintained asa separate element or integrated into the genome of the cells, as isknown in the art. A ribozyme-encoding DNA construct can includetranscriptional regulatory elements, such as a promoter element, anenhancer or UAS element, and a transcriptional terminator signal, forcontrolling transcription of ribozymes in the cells.

As taught in Haseloff et al., U.S. Pat. No. 5,641,673, ribozymes can beengineered so that ribozyme expression will occur in response to factorswhich induce expression of a target gene. Ribozymes also can beengineered to provide an additional level of regulation, so thatdestruction of mRNA occurs only when both a ribozyme and a target geneare induced in the cells.

Differentially Expressed Genes

Described herein are methods for the identification of genes whoseproducts interact with human prostasin-like enzyme. Such genes mayrepresent genes that are differentially expressed in disordersincluding, but not limited to, COPD, CNS disorders, and cancer. Further,such genes may represent genes that are differentially regulated inresponse to manipulations relevant to the progression or treatment ofsuch diseases. Additionally, such genes may have a temporally modulatedexpression, increased or decreased at different stages of tissue ororganism development. A differentially expressed gene may also have itsexpression modulated under control versus experimental conditions. Inaddition, the human prostasin-like enzyme gene or gene product mayitself be tested for differential expression.

The degree to which expression differs in a normal versus a diseasedstate need only be large enough to be visualized via standardcharacterization techniques such as differential display techniques.Other such standard characterization techniques by which expressiondifferences may be visualized include but are not limited to,quantitative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentially Expressed Genes

To identify differentially expressed genes total RNA or, preferably,mRNA is isolated from tissues of interest. For example, RNA samples areobtained from tissues of experimental subjects and from correspondingtissues of control subjects. Any RNA isolation technique that does notselect against the isolation of mRNA may be utilized for thepurification of such RNA samples. See, for example, Ausubel et al., ed.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. NewYork, 1987-1993. Large numbers of tissue samples may readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski,U.S. Pat. No. 4,843,155.

Transcripts within the collected RNA samples that represent RNA producedby differentially expressed genes are identified by methods well knownto those of skill in the art. They include, for example, differentialscreening (Tedder et al., Proc. Natl. Acad. Sci. U.S.A. 85, 208-12,1988), subtractive hybridization (Hedrick et al., Nature 308, 149-53;Lee et al., Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), anddifferential display (Liang & Pardee, Science 257, 967-71, 1992; U.S.Pat. No. 5,262,311), and microarrays.

The differential expression information may itself suggest relevantmethods for the treatment of disorders involving the humanprostasin-like enzyme. For example, treatment may include a modulationof expression of the differentially expressed genes and/or the geneencoding the human prostasin-like enzyme. The differential expressioninformation may indicate whether the expression or activity of thedifferentially expressed gene or gene product or the humanprostasin-like enzyme gene or gene product are up-regulated ordown-regulated.

Screening Methods

The invention provides assays for screening test compounds which bind toor modulate the activity of a human prostasin-like enzyme polypeptide ora human prostasin-like enzyme polynucleotide. A test compound preferablybinds to a human prostasin-like enzyme polypeptide or polynucleotide.More preferably, a test compound decreases or increases humanprostasin-like by at least about 10, preferably about 50, morepreferably about 75, 90, or 100% relative to the absence of the testcompound.

Test Compounds

Test compounds can be pharmacologic agents already known in the art orcan be compounds previously unknown to have any pharmacologicalactivity. The compounds can be naturally occurring or designed in thelaboratory. They can be isolated from microorganisms, animals, orplants, and can be produced recombinantly, or synthesized by chemicalmethods known in the art. If desired, test compounds can be obtainedusing any of the numerous combinatorial library methods known in theart, including but not limited to, biological libraries, spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibrary methods requiring deconvolution, the “one-bead one-compound”library method, and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide libraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer, or small molecule libraries ofcompounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in theart (see, for example, DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90,6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994;Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop etal., J. Med. Chem. 37, 1233, 1994). Libraries of compounds can bepresented in solution (see, e.g., Houghten, BioTechniques 13, 412-421,1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature364, 555-556, 1993), bacteria or spores (Ladner, U.S. Pat. No.5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. U.S.A. 89,1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990;Devlin, Science 249, 404-406, 1990); Cwirla et al., Proc. Natl. Acad.Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; andLadner, U.S. Pat. No. 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to humanprostasin-like enzyme polypeptides or polynucleotides or to affect humanprostasin-like enzyme activity or human prostasin-like enzyme geneexpression using high throughput screening. Using high throughputscreening, many discrete compounds can be tested in parallel so thatlarge numbers of test compounds can be quickly screened. The most widelyestablished techniques utilize 96-well microtiter plates. The wells ofthe microtiter plates typically require assay volumes that range from 50to 500 μl. In addition to the plates, many instruments, materials,pipettors, robotics, plate washers, and plate readers are commerciallyavailable to fit the 96-well format.

Alternatively, “free format assays,” or assays that have no physicalbarrier between samples, can be used. For example, an assay usingpigment cells (melanocytes) in a simple homogeneous assay forcombinatorial peptide libraries is described by Jayawickreme et al.,Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placedunder agarose in petri dishes, then beads that carry combinatorialcompounds are placed on the surface of the agarose. The combinatorialcompounds are partially released the compounds from the beads. Activecompounds can be visualized as dark pigment areas because, as thecompounds diffuse locally into the gel matrix, the active compoundscause the cells to change colors.

Another example of a free format assay is described by Chelsky,“Strategies for Screening Combinatorial Libraries: Novel and TraditionalApproaches,” reported at the First Annual Conference of The Society forBiomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelskyplaced a simple homogenous enzyme assay for carbonic anhydrase inside anagarose gel such that the enzyme in the gel would cause a color changethroughout the gel. Thereafter, beads carrying combinatorial compoundsvia a photolinker were placed inside the gel and the compounds werepartially released by UV-light. Compounds that inhibited the enzyme wereobserved as local zones of inhibition having less color change.

Yet another example is described by Salmon et al., Molecular Diversity2, 57-63 (1996). In this example, combinatorial libraries were screenedfor compounds that had cytotoxic effects on cancer cells growing inagar.

Another high throughput screening method is described in Beutel et al.,U.S. Pat. No. 5,976,813. In this method, test samples are placed in aporous matrix. One or more assay components are then placed within, ontop of, or at the bottom of a matrix such as a gel, a plastic sheet, afilter, or other form of easily manipulated solid support. When samplesare introduced to the porous matrix they diffuse sufficiently slowly,such that the assays can be performed without the test samples runningtogether.

Binding Assays

For binding assays, the test compound is preferably a small moleculewhich binds to and occupies, for example, the ATP/GTP binding site ofthe enzyme or the active site of the human prostasin-like enzymepolypeptide, such that normal biological activity is prevented. Examplesof such small molecules include, but are not limited to, small peptidesor peptide-like molecules.

In binding assays, either the test compound or the human prostasin-likeenzyme polypeptide can comprise a detectable label, such as afluorescent, radioisotopic, chemiluminescent, or enzymatic label, suchas horseradish peroxidase, alkaline phosphatase, or luciferase.Detection of a test compound which is bound to the human prostasin-likeenzyme polypeptide can then be accomplished, for example, by directcounting of radioemmission, by scintillation counting, or by determiningconversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to a human prostasin-likeenzyme polypeptide can be determined without labeling either of theinteractants. For example, a microphysiometer can be used to detectbinding of a test compound with a human prostasin-like enzymepolypeptide. A microphysiometer (e.g., Cytosensor™) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a test compound and a human prostasin-like enzymepolypeptide (McConnell et al., Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a humanprostasin-like enzyme polypeptide also can be accomplished using atechnology such as real-time Bimolecular Interaction Analysis (BIA)(Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo etal., Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technologyfor studying biospecific interactions in real time, without labeling anyof the interactants (e.g., BIAcore™). Changes in the optical phenomenonsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In yet another aspect of the invention, a human prostasin-like enzymepolypeptide can be used as a “bait protein” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.,Cell 72, 223-232, 1993; Madura et al., J. Biol. Chem. 268, 12046-12054,1993; Bartel et al., BioTechniques 14, 920-924, 1993; Iwabuchi et al.,Oncogene 8, 1693-1696, 1993; and Brent WO94/10300), to identify otherproteins which bind to or interact with the human prostasin-like enzymepolypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. For example, in one construct, polynucleotide encoding ahuman prostasin-like enzyme polypeptide can be fused to a polynucleotideencoding the DNA binding domain of a known transcription factor (e.g.,GAL-4). In the other construct a DNA sequence that encodes anunidentified protein (“prey” or “sample”) can be fused to apolynucleotide that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract in vivo to form an protein-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ), which is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected, and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the DNA sequenceencoding the protein which interacts with the human prostasin-likeenzyme polypeptide.

It may be desirable to immobilize either the human prostasin-like enzymepolypeptide (or polynucleotide) or the test compound to facilitateseparation of bound from unbound forms of one or both of theinteractants, as well as to accommodate automation of the assay. Thus,either the human prostasin-like enzyme polypeptide (or polynucleotide)or the test compound can be bound to a solid support. Suitable solidsupports include, but are not limited to, glass or plastic slides,tissue culture plates, microtiter wells, tubes, silicon chips, orparticles such as beads (including, but not limited to, latex,polystyrene, or glass beads). Any method known in the art can be used toattach the human prostasin-like enzyme polypeptide (or polynucleotide)or test compound to a solid support, including use of covalent andnon-covalent linkages, passive absorption, or pairs of binding moietiesattached respectively to the polypeptide (or polynucleotide) or testcompound and the solid support. Test compounds are preferably bound tothe solid support in an array, so that the location of individual testcompounds can be tracked. Binding of a test compound to a humanprostasin-like enzyme polypeptide (or polynucleotide) can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicrocentrifuge tubes.

In one embodiment, the human prostasin-like enzyme polypeptide is afusion protein comprising a domain that allows the human prostasin-likeenzyme polypeptide to be bound to a solid support. For example,glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and the non-adsorbed humanprostasin-like enzyme polypeptide; the mixture is then incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components.Binding of the interactants can be determined either directly orindirectly, as described above. Alternatively, the complexes can bedissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solidsupport also can be used in the screening assays of the invention. Forexample, either a human prostasin-like enzyme polypeptide (orpolynucleotide) or a test compound can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated humanprostasin-like enzyme polypeptides (or polynucleotides) or testcompounds can be prepared from biotin-NHS(N-hydroxysuccinimide) usingtechniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.) and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies which specifically bind to a human prostasin-like enzymepolypeptide, polynucleotide, or a test compound, but which do notinterfere with a desired binding site, such as the ATP/GTP binding siteor the active site of the human prostasin-like enzyme polypeptide, canbe derivatized to the wells of the plate. Unbound target or protein canbe trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies which specifically bind to the humanprostasin-like enzyme polypeptide or test compound, enzyme-linked assayswhich rely on detecting an activity of the human prostasin-like enzymepolypeptide, and SDS gel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to a human prostasin-like enzymepolypeptide or polynucleotide also can be carried out in an intact cell.Any cell which comprises a human prostasin-like enzyme polypeptide orpolynucleotide can be used in a cell-based assay system. A humanprostasin-like enzyme polynucleotide can be naturally occurring in thecell or can be introduced using techniques such as those describedabove. Binding of the test compound to a human prostasin-like enzymepolypeptide or polynucleotide is determined as described above.

Enzyme Assays

Test compounds can be tested for the ability to increase or decrease thehuman prostasin-like activity of a human prostasin-like enzymepolypeptide. Human prostasin-like enzyme activity can be measured, forexample, as described in Yu et al., J. Biol. Chem. 269, 18843-48, 1994.

Enzyme assays can be carried out after contacting either a purifiedhuman prostasin-like enzyme polypeptide, a cell membrane preparation, oran intact cell with a test compound. A test compound which decreases aserine protease activity of a human prostasin-like enzyme polypeptide byat least about 10, preferably about 50, more preferably about 75, 90, or100% is identified as a potential therapeutic agent for decreasing humanprostasin-like enzyme activity. A test compound which increases a serineprotease activity of a human prostasin-like enzyme polypeptide by atleast about 10, preferably about 50, more preferably about 75, 90, or100% is identified as a potential therapeutic agent for increasing humanprostasin-like enzyme activity.

Gene Expression

In another embodiment, test compounds which increase or decrease humanprostasin-like enzyme gene expression are identified. A humanprostasin-like enzyme polynucleotide is contacted with a test compound,and the expression of an RNA or polypeptide product of the humanprostasin-like enzyme polynucleotide is determined. The level ofexpression of appropriate mRNA or polypeptide in the presence of thetest compound is compared to the level of expression of mRNA orpolypeptide in the absence of the test compound. The test compound canthen be identified as a modulator of expression based on thiscomparison. For example, when expression of mRNA or polypeptide isgreater in the presence of the test compound than in its absence, thetest compound is identified as a stimulator or enhancer of the mRNA orpolypeptide expression. Alternatively, when expression of the mRNA orpolypeptide is less in the presence of the test compound than in itsabsence, the test compound is identified as an inhibitor of the mRNA orpolypeptide expression.

The level of human prostasin-like enzyme mRNA or polypeptide expressionin the cells can be determined by methods well known in the art fordetecting mRNA or polypeptide. Either qualitative or quantitativemethods can be used. The presence of polypeptide products of a humanprostasin-like enzyme polynucleotide can be determined, for example,using a variety of techniques known in the art, including immunochemicalmethods such as radioimmunoassay, Western blotting, andimmunohistochemistry. Alternatively, polypeptide synthesis can bedetermined in vivo, in a cell culture, or in an in vitro translationsystem by detecting incorporation of labeled amino acids into a humanprostasin-like enzyme polypeptide.

Such screening can be carried out either in a cell-free assay system orin an intact cell. Any cell which expresses a human prostasin-likeenzyme polynucleotide can be used in a cell-based assay system. Thehuman prostasin-like enzyme polynucleotide can be naturally occurring inthe cell or can be introduced using techniques such as those describedabove. Either a primary culture or an established cell line, such as CHOor human embryonic kidney 293 cells, can be used.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions which can beadministered to a patient to achieve a therapeutic effect.Pharmaceutical compositions of the invention can comprise aprostasin-like enzyme polypeptide, prostasin-like enzyme polynucleotide,antibodies which specifically bind to a prostasin-like enzymepolypeptide, or mimetics, agonists, antagonists, or inhibitors of aprostasin-like enzyme polypeptide. The compositions can be administeredalone or in combination with at least one other agent, such asstabilizing compound, which can be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions can beadministered to a patient alone, or in combination with other agents,drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositionscan contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically.Pharmaceutical compositions of the invention can be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, parenteral, topical, sublingual, or rectal means.Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores can be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which also can contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments can be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds canbe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration canbe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions cancontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds can be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers also can be used for delivery. Optionally, the suspensionalso can contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. For topical or nasal administration, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition can be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation can be alyophilized powder which can contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can befound in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES(Maack Publishing Co., Easton, Pa.). After pharmaceutical compositionshave been prepared, they can be placed in an appropriate container andlabeled for treatment of an indicated condition. Such labeling wouldinclude amount, frequency, and method of administration.

Therapeutic Indications and Methods

1. Tumor Cell Invasion and Metastasis.

The human prostasin-like enzyme gene provides a therapeutic target fordecreasing human prostasin-like enzyme activity, in particular fortreating or preventing metastatic cancer. Cancer is a diseasefundamentally caused by oncogenic cellular transformation. There areseveral hallmarks of transformed cells that distinguish them from theirnormal counterparts and underlie the pathophysiology of cancer. Theseinclude uncontrolled cellular proliferation, unresponsiveness to normaldeath-inducing signals (immortalization), increased cellular motilityand invasiveness, increased ability to recruit blood supply throughinduction of new blood vessel formation (angiogenesis), geneticinstability, and dysregulated gene expression. Various combinations ofthese aberrant physiologies, along with the acquisition ofdrug-resistance frequently lead to an intractable disease state in whichorgan failure and patient death ultimately ensue.

Most standard cancer therapies target cellular proliferation and rely onthe differential proliferative capacities between transformed and normalcells for their efficacy. This approach is hindered by the facts thatseveral important normal cell types are also highly proliferative andthat cancer cells frequently become resistant to these agents. Thus, thetherapeutic indices for traditional anti-cancer therapies rarely exceed2.0.

The advent of genomics-driven molecular target identification has openedup the possibility of identifying new cancer-specific targets fortherapeutic intervention that will provide safer, more effectivetreatments for cancer patients. Thus, newly discovered tumor-associatedgenes and their products can be tested for their role(s) in disease andused as tools to discover and develop innovative therapies. Genesplaying important roles in any of the physiological processes outlinedabove can be characterized as cancer targets.

Genes or gene fragments identified through genomics can readily beexpressed in one or more heterologous expression systems to producefunctional recombinant proteins. These proteins are characterized invitro for their biochemical properties and then used as tools inhigh-throughput molecular screening programs to identify chemicalmodulators of their biochemical activities. Agonists and/or antagonistsof target protein activity can be identified in this manner andsubsequently tested in cellular and in vivo disease models foranti-cancer activity. Optimization of lead compounds with iterativetesting in biological models and detailed pharmacokinetic andtoxicological analyses form the basis for drug development andsubsequent testing in humans.

For example, blocking a fibronectin domain of human prostasin-likeenzyme can suppress or prevent migration or metastasis of tumor cells inresponse to fibronectin (9, 10). Cancers whose metastasis can besuppressed according to the invention include adenocarcinoma, melanoma,cancers of the adrenal gland, bladder, bone, breast, cervix, gallbladder, liver, lung, ovary, pancreas, prostate, testis, and uterus.Circulating tumor cells arrested in the capillary beds of differentorgans must invade the endothelial cell lining and degrade itsunderlying basement membrane (BM) in order to invade into theextravascular tissue(s) where they establish metastasis (1, 2).Metastatic tumor cells often attach at or near the intercellularjunctions between adjacent endothelial cells. Such attachment of themetastatic cells is followed by rupture of the junctions, retraction ofthe endothelial cell borders and migration through the breach in theendothelium toward the exposed underlying BM (1, 11).

Once located between endothelial cells and the BM, the invading cellsmust degrade the subendothelial glycoproteins and proteoglycans of theBM in order to migrate out of the vascular compartment. Several cellularenzymes (e.g., collagenase IV, plasminogen activator, cathepsin B,elastase) are thought to be involved in degradation of BM (2, 11).Suppression of human prostasin-like enzyme activity therefore can beused to suppress tumor cell invasion and metastasis.

2. Tumor Angiogenesis.

Basic fibroblast growth factor (bFGF) has been extracted from thesubendothelial extracellular matrix produced in vitro (3) and frombasement membranes of the cornea (4), suggesting that extracellularmatrix may serve as a reservoir for bFGF. Immunohistochemical stainingrevealed the localization of bFGF in basement membranes of diversetissues and blood vessels (5). Despite the ubiquitous presence of bFGFin normal tissues, endothelial cell proliferation in these tissues isusually very low, which suggests that bFGF is somehow sequestered fromits site of action. It is possible, therefore, that suppression of humanprostasin-like enzyme activity can suppress release of active bFGF fromextracellular matrix and basement membranes. In addition, displacementof bFGF from its storage within basement membranes and extracellularmatrix may therefore provide a novel mechanism for induction ofneovascularization in normal and pathological situations. Restriction ofendothelial cell growth factors in the extracellular matrix may preventtheir systemic action on the vascular endothelium, thus maintaining avery low rate of endothelial cells turnover and vessel growth. On theother hand, release of bFGF from storage in the extracellular matrix mayelicit localized endothelial cell proliferation and neovascularizationin processes such as wound healing, inflammation and tumor development(6, 7).

3. Inflammation and Cellular Immunity.

Prostasin-like enzyme activity may be involved in the ability ofactivated cells of the immune system to leave the circulation and elicitboth inflammatory and autoimmune responses. Thus, inflammation andcellular immunity may be regulated by regulating activity ofprostasin-like enzyme.

4. Viral Infection.

Removal of the cell surface components by prostasin-like enzyme mayinfluence the ability of viruses to attach to the cell surface.Regulation of prostasin-like enzyme may therefore be used to treat viralinfections.

5. Neurodegenerative Diseases.

It is also possible that prostasin-like enzyme activity can be used todegrade, for example, prion protein amyloid plaques ofGenstmann-Straussler Syndrome, Creutzfeldt-Jakob disease, and Scrapie.

6. Restenosis and Atherosclerosis.

Proliferation of arterial smooth muscle cells (SMCs) in response toendothelial injury and accumulation of cholesterol rich lipoproteins arebasic events in the pathogenesis of atherosclerosis and restenosis (8).It is possible that prostasin-like enzyme may be involved in thecatabolic pathway that may allow substantial cellular and interstitialaccumulation of cholesterol rich lipoproteins. The latter pathway isexpected to be highly atherogenic by promoting accumulation of apoB andapoE rich lipoproteins (i.e. LDL, VLDL, chylomicrons), independent offeedback inhibition by the cellular sterol content. Altered levels ofhuman prostasin-like enzyme activity therefore may inhibit both SMCproliferation and lipid accumulation and thus may halt the progressionof restenosis and atherosclerosis.

7. Osteoporosis.

Osteoporosis is a disease characterized by low bone mass andmicroarchitectural deterioration of bone tissue, leading to enhancedbone fragility and a consequent increase in fracture risk. It is themost common human metabolic bone disorder. Established osteoporosisincludes the presence of fractures.

Bone turnover occurs by the action of two major effector cell typeswithin bone: the osteoclast, which is responsible for bone resorption,and the osteoblast, which synthesizes and mineralizes bone matrix. Theactions of osteoclasts and osteoblasts are highly coordinated.Osteoclast precursors are recruited to the site of turnover; theydifferentiate and fuse to form mature osteoclasts which then resorbbone. Attached to the bone surface, osteoclasts produce an acidicmicroenvironment in a tightly defined junction between the specializedosteoclast border membrane and the bone matrix, thus allowing thelocalized solubilization of bone matrix. This in turn facilitate theproteolysis of demineralized bone collagen. Matrix degradation isthought to release matrix-associated growth factor and cytokines, whichrecruit osteoblasts in a temporally and spatially controlled fashion.Osteoblasts synthesize and secrete new bone matrix proteins, andsubsequently mineralize this new matrix. In the normal skeleton this isa physiological process which does not result in a net change in bonemass. In pathological states, such as osteoporosis, the balance betweenresorption and formation is altered such that bone loss occurs. See WO99/45923.

The osteoclast itself is the direct or indirect target of all currentlyavailable osteoporosis agents with the possible exception of fluoride.Antiresorptive therapy prevents further bone loss in treatedindividuals. Osteoblasts are derived from multipotent stem cells whichreside in bone marrow and also gives rise to adipocytes, chondrocytes,fibroblasts and muscle cells. Selective enhancement of osteoblastactivity is a highly desirable goal for osteoporosis therapy since itwould result in an increase in bone mass, rather than a prevention offurther bone loss. An effective anabolic therapy would be expected tolead to a significantly greater reduction in fracture risk thancurrently available treatments.

The agonists or antagonists to the newly discovered polypeptides may actas antiresorptive by directly altering the osteoclast differentiation,osteoclast adhesion to the bone matrix or osteoclast function ofdegrading the bone matrix. The agonists or antagonists could indirectlyalter the osteoclast function by interfering in the synthesis and/ormodification of effector molecules of osteoclast differentiation orfunction such as cytokines, peptide or steroid hormones, proteases, etc.

The agonists or antagonists to the newly discovered polypeptides may actas anabolics by directly enhancing the osteoblast differentiation and/orits bone matrix forming function. The agonists or antagonists could alsoindirectly alter the osteoblast function by enhancing the synthesis ofgrowth factors, peptide or steroid hormones or decreasing the synthesisof inhibitory molecules.

The agonists and antagonists may be used to mimic, augment or inhibitthe action of the newly discovered polypeptides which may be useful totreat osteoporosis, Paget's disease, degradation of bone implantsparticularly dental implants.

8. COPD.

Chronic obstructive pulmonary (or airways) disease (COPD) is a conditiondefined physiologically as airflow obstruction that generally resultsfrom a mixture of emphysema and peripheral airway obstruction due tochronic bronchitis (Senior & Shapiro, Pulmonary Diseases and Disorders,3d ed., New York, McGraw-Hill, 1998, pp. 659-681, 1998; Barnes, Chest117, 10S-14S, 2000). Emphysema is characterized by destruction ofalveolar walls leading to abnormal enlargement of the air spaces of thelung. Chronic bronchitis is defined clinically as the presence ofchronic productive cough for three months in each of two successiveyears. In COPD, airflow obstruction is usually progressive and is onlypartially reversible. By far the most important risk factor fordevelopment of COPD is cigarette smoking, although the disease doesoccur in non-smokers.

Chronic inflammation of the airways is a key pathological feature ofCOPD (Senior & Shapiro, 1998). The inflammatory cell populationcomprises increased numbers of macrophages, neutrophils, and CD8⁺lymphocytes. Inhaled irritants, such as cigarette smoke, activatemacrophages which are resident in the respiratory tract, as well asepithelial cells leading to release of chemokines (e.g., interleukin-8)and other chemotactic factors. These chemotactic factors act to increasethe neutrophil/monocyte trafficking from the blood into the lung tissueand airways. Neutrophils and monocytes recruited into the airways canrelease a variety of potentially damaging mediators such as proteolyticenzymes and reactive oxygen species. Matrix degradation and emphysema,along with airway wall thickening, surfactant dysfunction, and mucushypersecretion, all are potential sequelae of this inflammatory responsethat lead to impaired airflow and gas exchange.

COPD is characterized by damage to the lung extracellular matrix andemphysema can be viewed as the pathologic process that affects the lungparenchyma. This process eventually leads to the destruction of theairway walls resulting in permanent airspace enlargement (Senior andShapiro, in PULMONARY DISEASES AND DISORDERS, 3rd ed., New York,McGraw-Hill, 1998, pp. 659-681, 1998). The observation that inheriteddeficiency of a1-antitrypsin (a1-AT), the primary inhibitor ofneutrophil elastase, predisposes individuals to early onset emphysema,and that intrapulmonary instillation of elastolytic enzymes inexperimental animals causes emphysema, led to the elastase:antielastasehypothesis for the pathogenesis of emphysema (Eriksson, Acta Med. Scand.177 (Suppl.), 432, 1965, Gross, J. Occup. Med. 6, 481-84, 1964). This inturn led to the concept that destruction of elastin in the lungparenchyma is the basis of the development of emphysema.

A broad range of immune and inflammatory cells including neutrophils,macrophages, T lymphocytes and eosinophils contain proteolytic enzymesthat could contribute to the destruction of lung extracellular matrix(Shapiro, 1999). In addition, a number of different classes of proteaseshave been identified that have the potential to contribute to lungmatrix destruction. These include serine proteases, matrixmetalloproteinases and cysteine proteases. Of these classes of enzymes,a number can hydrolyze elastin and have been shown to be elevated inCOPD patients (neutrophil elastase, MMP-2, 9, 12) (Culpitt et al., Am.J. Respir. Crit. Care Med. 160, 1635-39, 1999, Shapiro, Am. J. Crit.Care Med. 160 (5), S29-S32, 1999).

It is expected that in the future novel members of the existing classesof proteases and new classes of proteases will be identified that play asignificant role in the damage of the extracellular lung matrixincluding elastin proteolysis. Novel protease targets therefore remainvery attractive therapeutic targets.

9. Other Therapeutic and Diagnostic Indications.

Anti-human prostasin-like enzyme antibodies can be applied forimmunodetection and diagnosis of micrometastases, autoimmune lesions,and renal failure in biopsy specimens, plasma samples, and body fluids.Alternatively, if desired a prostasin-like enzyme function can besupplied to a cell by introducing a prostasin-like enzyme-encodingpolynucleotide into the cell.

The invention further pertains to the use of novel agents identified bythe screening assays described above. Accordingly, it is within thescope of this invention to use a test compound identified as describedherein in an appropriate animal model. For example, an agent identifiedas described herein (e.g., a modulating agent, an antisense nucleic acidmolecule, a specific antibody, ribozyme, or a polypeptide-bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

A reagent which affects prostasin-like enzyme activity can beadministered to a human cell, either in vitro or in vivo, to reduceprostasin-like enzyme activity. The reagent preferably binds to anexpression product of a human prostasin-like enzyme gene. If theexpression product is a polypeptide, the reagent is preferably anantibody. For treatment of human cells ex vivo, an antibody can be addedto a preparation of stem cells which have been removed from the body.The cells can then be replaced in the same or another human body, withor without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome.Preferably, the liposome is stable in the animal into which it has beenadministered for at least about 30 minutes, more preferably for at leastabout 1 hour, and even more preferably for at least about 24 hours. Aliposome comprises a lipid composition that is capable of targeting areagent, particularly a polynucleotide, to a particular site in ananimal, such as a human. Preferably, the lipid composition of theliposome is capable of targeting to a specific organ of an animal, suchas the lung or liver.

A liposome useful in the present invention comprises a lipid compositionthat is capable of fusing with the plasma membrane of the targeted cellto deliver its contents to the cell. Preferably, the transfectionefficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposomedelivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16mmol of liposome delivered to about 10⁶ cells, and even more preferablyabout 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶cells. Preferably, a liposome is between about 100 and 500 nm, morepreferably between about 150 and 450 nm, and even more preferablybetween about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include thoseliposomes standardly used in, for example, gene delivery methods knownto those of skill in the art. More preferred liposomes include liposomeshaving a polycationic lipid composition and/or liposomes having acholesterol backbone conjugated to polyethylene glycol. Optionally, aliposome comprises a compound capable of targeting the liposome to atumor cell, such as a tumor cell ligand exposed on the outer surface ofthe liposome.

Complexing a liposome with a reagent such as an antisenseoligonucleotide or ribozyme can be achieved using methods which arestandard in the art (see, for example, U.S. Pat. No. 5,705,151).Preferably, from about 0.1 μg to about 10 μg of polynucleotide iscombined with about 8 nmol of liposomes, more preferably from about 0.5μg to about 5 μg of polynucleotides are combined with about 8 nmolliposomes, and even more preferably about 1.0 μg of polynucleotides iscombined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissuesin vivo using receptor-mediated targeted delivery. Receptor-mediated DNAdelivery techniques are taught in, for example, Findeis et al. Trends inBiotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODSAND APPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.) (1994); Wu &Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269,542-46 (1994); Zenke et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59(1990); Wu et al., J. Biol. Chem. 266, 338-42 (1991).

If the reagent is a single-chain antibody, polynucleotides encoding theantibody can be constructed and introduced into a cell either ex vivo orin vivo using well-established techniques including, but not limited to,transferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun,” and DEAE- orcalcium phosphate-mediated transfection.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within thecapability of those skilled in the art. A therapeutically effective doserefers to that amount of active ingredient which increases or decreaseshuman prostasin-like enzyme activity relative to that which occurs inthe absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model also can be used todetermine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeuticallyeffective in 50% of the population) and LD₅₀ (the dose lethal to 50% ofthe population), can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors which can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions can be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Effective in vivo dosages of an antibody are in the range of about 5 μgto about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about500 μg/kg of patient body weight, and about 200 to about 250 μg/kg ofpatient body weight. For administration of polynucleotides encodingsingle-chain antibodies, effective in vivo dosages are in the range ofabout 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg ofDNA.

If the expression product is mRNA, the reagent is preferably anantisense oligonucleotide or a ribozyme. Polynucleotides which expressantisense oligonucleotides or ribozymes can be introduced into cells bya variety of methods, as described above.

Preferably, a reagent reduces expression of a prostasin-like enzymepolynucleotide or activity of a prostasin-like enzyme polypeptide by atleast about 10, preferably about 50, more preferably about 75, 90, or100% relative to the absence of the reagent. The effectiveness of themechanism chosen to decrease the level of expression of a prostasin-likeenzyme polynucleotide or the activity of a prostasin-like enzymepolypeptide can be assessed using methods well known in the art, such ashybridization of nucleotide probes to prostasin-like enzyme-specificmRNA, quantitative RT-PCR, immunologic detection of a prostasin-likeenzyme polypeptide, or measurement of prostasin-like enzyme activity.

In any of the embodiments described above, any of the pharmaceuticalcompositions of the invention can be administered in combination withother appropriate therapeutic agents. Selection of the appropriateagents for use in combination therapy can be made by one of ordinaryskill in the art, according to conventional pharmaceutical principles.The combination of therapeutic agents can act synergistically to effectthe treatment or prevention of the various disorders described above.Using this approach, one may be able to achieve therapeutic efficacywith lower dosages of each agent, thus reducing the potential foradverse side effects.

Any of the therapeutic methods described above can be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

The above disclosure generally describes the present invention, and allpatents and patent applications cited in this disclosure are expresslyincorporated herein. A more complete understanding can be obtained byreference to the following specific examples which are provided forpurposes of illustration only and are not intended to limit the scope ofthe invention.

EXAMPLE 1 Detection of Prostasin-Like Enzyme Activity

The polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 5 is inserted into theexpression vector pCEV4 and the expression vector pCEV4-prostasin-likeenzyme polypeptide obtained is transfected into human embryonic kidney293 cells. From these cells extracts are obtained and protease activityis measured using thiobenzylester substrates, as described in U.S. Pat.No. 5,500,344. For monitoring enzyme activities from granules and columnfractions, assays are performed at room temperature using 0.5 mM5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (Sigma) to detect the HSBzlleaving group (₄₁₀=13600 M⁻¹ cm⁻¹).

BLT-esterase activity is estimated using a microtiter assay (Green andShaw, Anal. Biochem. 93, 223-226, 1979). Briefly, 50 μl of sample isadded to 100 μl of 1 mM DTNB, made up in 10 mM HEPES, 1 mM CaCl₂, 1 mMMgCl₂, pH 7.2. The reaction is initiated by the addition of 50 μl of BLT(Sigma) to give a final concentration of 500 μM. For Metasedeterminations, 50 μl of dilutions of the sample in 0.1 M HEPES, 0.05 MCaCl₂, pH 7.5, are added to 100 μl of 1 mM DTNB, and the reaction isinitiated by the addition of 50 μl of Boc-Ala-Ala-Met-S Benzyl (Bzl) togive a final concentration of 150 μM. The duration of the assay dependson color development, the rate of which is measured (O.D.₄₁₀) on aDynatech MR 5000 microplate reader. Controls of sample and DTNB alone orDTNB and substrate alone are run.

For more sensitive comparisons of enzymatic activities, peptidethiobenzyl ester substrates are used to measure protease activities. Thechymase substrate Suc-Phe-Leu-Phe-SBzl is purchased from BACHEMBioscience Inc., Philadelphia, Pa. Z-Arg-SBzl (the tryptase substrate,Kam et al., J. Biol. Chem. 262, 3444-3451, 1987); Boc-Ala-Ala-AA-SBzl(AA=Asp, Met, Leu, Nle, or Ser), and Suc-Ala-Ala-Met-SBzl (Odake et al,Biochemistry 30, 2217-2227, 1991); Harper et al., Biochemistry 23,2995-3002, 1984) are synthesized previously. Boc-Ala-Ala-Asp-SBzl is thesubstrate for Asp-ase and peptide thiobenzyl esters containing Met, Leuor Nle are substrates for Met-ase SP. Assays are performed at roomtemperature in 0.1 M, HEPES buffer, pH 7.5, containing 0.01 M CaCl₂ and8% Me₂O using 0.34 mM 4,4′-dithiodipyridine (Aldrithiol-4, AldrichChemical Co., Milwaukee, Wis.) to detect HSBzl leaving group that reactswith 4,4′-dithiodipyridine to release thiopyridone (324=19800 M⁻¹ cm⁻¹,Grasetti and Murray, Arch. Biochem. Biophys. 119, 41-49, 1967). Theinitial rates are measured at 324 nm using a Beckman 35spectro-photometer when 10-25 μl of an enzyme stock solution is added toa cuvette containing 2.0 ml of buffer, 150 μl of 4,4′-dithiodipyridine,and 25 μl of substrate. The same volume of substrate and4,4′-dithiodipyridine are added to the reference cell in order tocompensate for the background hydrolysis rate of the substrates. Initialrates are measured in duplicate for each substrate concentration and areaveraged in each case. Substrate concentrations are 100-133 μM. It isshown that the polypeptide of SEQ ID NO: 2 and or SEQ ID NO: 6 haveprostasin-like enzyme activity.

EXAMPLE 2 Identification of a Test Compound which Binds to aProstasin-Like Enzyme Polypeptide

Purified prostasin-like enzyme polypeptides comprising aglutathione-S-transferase protein and absorbed ontoglutathione-derivatized wells of 96-well microtiter plates are contactedwith test compounds from a small molecule library at pH 7.0 in aphysiological buffer solution. Prostasin-like enzyme polypeptidescomprise the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO: 6.The test compounds comprise a fluorescent tag. The samples are incubatedfor 5 minutes to one hour. Control samples are incubated in the absenceof a test compound.

The buffer solution containing the test compounds is washed from thewells. Binding of a test compound to a prostasin-like enzyme polypeptideis detected by fluorescence measurements of the contents of the wells. Atest compound which increases the fluorescence in a well by at least 15%relative to fluorescence of a well in which a test compound was notincubated is identified as a compound which binds to a prostasin-likeenzyme polypeptide.

EXAMPLE 3 Identification of a Test Compound which DecreasesProstasin-Like Enzyme Activity

Cellular extracts from the human colon cancer cell line HCT116 arecontacted with test compounds from a small molecule library and assayedfor prostasin-like enzyme activity. Control extracts, in the absence ofa test compound, also are assayed. Protease activity can be measuredusing thiobenzylester substrates, as described in U.S. Pat. No.5,500,344. For monitoring enzyme activities from granules and columnfractions, assays are performed at room temperature using 0.5 mM5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (Sigma) to detect the HSBzlleaving group (₄₁₀=13600 M⁻¹ cm⁻¹).

BLT-esterase activity is estimated using a microtiter assay (Green andShaw, Anal. Biochem. 93, 223-226, 1979). Briefly, 50 μl of sample isadded to 100 μl of 1 mM DTNB, made up in 10 mM HEPES, 1 mM CaCl₂, 1 mMMgCl₂, pH 7.2. The reaction is initiated by the addition of 50 μl of BLT(Sigma) to give a final concentration of 500 μM. For Metasedeterminations, 50 μl of dilutions of the sample in 0.1 M HEPES, 0.05 MCaCl₂, pH 7.5, are added to 100 μl of 1 mM DTNB, and the reaction isinitiated by the addition of 50 μl of Boc-Ala-Ala-Met-S Benzyl (Bzl) togive a final concentration of 150 μM. The duration of the assay dependson color development, the rate of which is measured (O.D.₄₁₀) on aDynatech MR 5000 microplate reader. Controls of sample and DTNB alone orDTNB and substrate alone are run.

For more sensitive comparisons of enzymatic activities, peptidethiobenzyl ester substrates are used to measure protease activities. Thechymase substrate Suc-Phe-Leu-Phe-SBzl is purchased from BACHEMBioscience Inc., Philadelphia, Pa. Z-Arg-SBzl (the tryptase substrate,Kam et al., J. Biol. Chem. 262, 3444-3451, 1987); Boc-Ala-Ala-AA-SBzl(AA=Asp, Met, Leu, Nle, or Ser), and Suc-Ala-Ala-Met-SBzl (Odake et al.,Biochemistry 30, 2217-2227, 1991); Harper et al., Biochemistry 23,2995-3002, 1984) are synthesized previously. Boc-Ala-Ala-Asp-SBzl is thesubstrate for Asp-ase and peptide thiobenzyl esters containing Met, Leuor Nle are substrates for Met-ase SP. Assays are performed at roomtemperature in 0.1 M, HEPES buffer, pH 7.5, containing 0.01 M CaCl₂ and8% Me₂O using 0.34 mM 4,4′-dithiodipyridine (Aldrithiol-4, AldrichChemical Co., Milwaukee, Wis.) to detect HSBzl leaving group that reactswith 4,4′-dithiodipyridine to release thiopyridone (324=19800 M⁻¹ cm⁻¹,Grasetti and Murray, Arch. Biochem. Biophys. 119, 41-49, 1967). Theinitial rates are measured at 324 nm using a Beckman 35spectrophotometer when 10-25 μl of an enzyme stock solution is added toa cuvette containing 2.0 ml of buffer, 150 μl of 4,4′-dithiodipyridine,and 25 μl of substrate. The same volume of substrate and4,4′-dithiodipyridine are added to the reference cell in order tocompensate for the background hydrolysis rate of the substrates. Initialrates are measured in duplicate for each substrate concentration and areaveraged in each case. Substrate concentrations are 100-133 μM.

Alternatively, prostasin activity can be measured as described in Yu etal., J. Biol. Chem. 269, 11843-48, 1994.

A test compound which decreases prostasin-like enzyme activity of theextract relative to the control extract by at least 20% is identified asa prostasin-like enzyme inhibitor.

EXAMPLE 4 Identification of a Test Compound which DecreasesProstasin-Like Enzyme Gene Expression

A test compound is administered to a culture of the breast tumor cellline MDA-468 and incubated at 37° C. for 10 to 45 minutes. A culture ofthe same type of cells incubated for the same time without the testcompound provides a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al.,Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30μg total RNA and hybridized with a ³²P-labeled prostasin-likeenzyme-specific probe at 65° C. in Express-hyb (CLONTECH). The probecomprises at least 11 contiguous nucleotides selected from thecomplement of SEQ ID NO:1 or SEQ ID NO: 5. A test compound whichdecreases the prostasin-like enzyme-specific signal relative to thesignal obtained in the absence of the test compound is identified as aninhibitor of prostasin-like enzyme gene expression.

EXAMPLE 5 Treatment of a Breast Tumor with a Reagent which SpecificallyBinds to a Prostasin-Like Enzyme Gene Product

Synthesis of antisense prostasin-like enzyme oligonucleotides comprisingat least 11 contiguous nucleotides selected from the complement of SEQID NO:1 or SEQ ID NO: 5 is performed on a Pharmacia Gene Assemblerseries synthesizer using the phosphoramidite procedure (Uhlmann et al.,Chem. Rev. 90, 534-83, 1990). Following assembly and deprotection,oligonucleotides are ethanol-precipitated twice, dried, and suspended inphosphate-buffered saline (PBS) at the desired concentration. Purity ofthese oligonucleotides is tested by capillary gel electrophoreses andion exchange HPLC. Endotoxin levels in the oligonucleotide preparationare determined using the Limulus Amebocyte Assay (Bang, Biol. Bull.(Woods Hole, Mass.) 105, 361-362, 1953).

An aqueous composition containing the antisense oligonucleotides at aconcentration of 0.1-100 μM is injected directly into a breast tumorwith a needle. The needle is placed in the tumors and withdrawn whileexpressing the aqueous composition within the tumor.

The breast tumor is monitored over a period of days or weeks. Additionalinjections of the antisense oligonucleotides can be given during thattime. Metastasis of the breast tumor is suppressed due to decreasedprostasin-like enzyme activity of the breast tumor cells.

EXAMPLE 6 Expression of Recombinant Human Prostasin-Like Serine Protease

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego,Calif.) is used to produce large quantities of recombinant humanprostasin-like serine protease polypeptides in yeast. The prostasin-likeserine protease-encoding DNA sequence is derived from SEQ ID NO:1 or SEQID NO: 5. Before insertion into vector pPICZB, the DNA sequence ismodified by well known methods in such a way that it contains at its5′-end an initiation codon and at its 3′-end an enterokinase cleavagesite, a His6 reporter tag and a termination codon. Moreover, at bothtermini recognition sequences for restriction endonucleases are addedand after digestion of the multiple cloning site of pPICZ B with thecorresponding restriction enzymes the modified DNA sequence is ligatedinto pPICZB. This expression vector is designed for inducible expressionin Pichia pastoris, driven by a yeast promoter. The resultingpPICZ/md-His6 vector is used to transform the yeast.

The yeast is cultivated under usual conditions in 5 liter shake flasksand the recombinantly produced protein isolated from the culture byaffinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. Thebound polypeptide is eluted with buffer, pH 3.5, and neutralized.Separation of the polypeptide from the His6 reporter tag is accomplishedby site-specific proteolysis using enterokinase (Invitrogen, San Diego,Calif.) according to manufacturer's instructions. Purified humanprostasin-like serine protease polypeptide is obtained.

EXAMPLE 7 In Vivo Testing of Compounds/Target Validation

1. Acute Mechanistic Assays

1.1. Reduction in Mitogenic Plasma Hormone Levels

This non-tumor assay measures the ability of a compound to reduce eitherthe endogenous level of a circulating hormone or the level of hormoneproduced in response to a biologic stimulus. Rodents are administeredtest compound (p.o., i.p., i.v., i.m., or s.c.). At a predetermined timeafter administration of test compound, blood plasma is collected. Plasmais assayed for levels of the hormone of interest. If the normalcirculating levels of the hormone are too low and/or variable to provideconsistent results, the level of the hormone may be elevated by apre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m.into mice at a dosage of 30 ng/mouse to induce a burst of testosteronesynthesis). The timing of plasma collection would be adjusted tocoincide with the peak of the induced hormone response. Compound effectsare compared to a vehicle-treated control group. An F-test is preformedto determine if the variance is equal or unequal followed by a Student'st-test. Significance is p value≦0.05 compared to the vehicle controlgroup.

1.2. Hollow Fiber Mechanism of Action Assay

Hollow fibers are prepared with desired cell line(s) and implantedintraperitoneally and/or subcutaneously in rodents. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol, these may includeassays for gene expression (bDNA, PCR, or Taqman), or a specificbiochemical activity (i.e., cAMP levels. Results are analyzed byStudent's t-test or Rank Sum test after the variance between groups iscompared by an F-test, with significance at p≦0.05 as compared to thevehicle control group.

2. Subacute Functional in Vivo Assays

2.1. Reduction in Mass of Hormone Dependent Tissues

This is another non-tumor assay that measures the ability of a compoundto reduce the mass of a hormone dependent tissue (i.e., seminal vesiclesin males and uteri in females). Rodents are administered test compound(p.o., i.p., i.v., i.m., or s.c.) according to a predetermined scheduleand for a predetermined duration (i.e., 1 week). At termination of thestudy, animals are weighed, the target organ is excised, any fluid isexpressed, and the weight of the organ is recorded. Blood plasma mayalso be collected. Plasma may be assayed for levels of a hormone ofinterest or for levels of test agent. Organ weights may be directlycompared or they may be normalized for the body weight of the animal.Compound effects are compared to a vehicle-treated control group. AnF-test is preformed to determine if the variance is equal or unequalfollowed by a Student's t-test. Significance is p value≦0.05 compared tothe vehicle control group.

2.2. Hollow Fiber Proliferation Assay

Hollow fibers are prepared with desired cell line(s) and implantedintraperitoneally and/or subcutaneously in rodents. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol. Cell proliferation isdetermined by measuring a marker of cell number (i.e., MTT or LDH). Thecell number and change in cell number from the starting inoculum areanalyzed by Student's t-test or Rank Sum test after the variance betweengroups is compared by an F-test, with significance at p≦0.05 as comparedto the vehicle control group.

2.3. Anti-Angiogenesis Models

2.3.1. Corneal Angiogenesis

Hydron pellets with or without growth factors or cells are implantedinto a micropocket surgically created in the rodent cornea. Compoundadministration may be systemic or local (compound mixed with growthfactors in the hydron pellet). Corneas are harvested at 7 days postimplantation immediately following intracardiac infusion of colloidalcarbon and are fixed in 10% formalin. Readout is qualitative scoringand/or image analysis. Qualitative scores are compared by Rank Sum test.Image analysis data is evaluated by measuring the area ofneovascularization (in pixels) and group averages are compared byStudent's t-test (2 tail). Significance is p≦0.05 as compared to thegrowth factor or cells only group.

2.3.2. Matrigel Angiogenesis

Matrigel, containing cells or growth factors, is injectedsubcutaneously. Compounds are administered p.o., i.p., i.v., i.m., ors.c. Matrigel plugs are harvested at predetermined time point(s) andprepared for readout. Readout is an ELISA-based assay for hemoglobinconcentration and/or histological examination (i.e. vessel count,special staining for endothelial surface markers: CD31, factor-8).Readouts are analyzed by Student's t-test, after the variance betweengroups is compared by an F-test, with significance determined at p≦0.05as compared to the vehicle control group.

3. Primary Antitumor Efficacy

3.1. Early Therapy Models

3.1.1. Subcutaneous Tumor

Tumor cells or fragments are implanted subcutaneously on Day 0. Vehicleand/or compounds are administered p.o., i.p., i.v., i.m., or s.c.according to a predetermined schedule starting at a time, usually on Day1, prior to the ability to measure the tumor burden. Body weights andtumor measurements are recorded 2-3 times weekly. Mean net body andtumor weights are calculated for each data collection day. Anti-tumorefficacy may be initially determined by comparing the size of treated(T) and control (C) tumors on a given day by a Student's t-test, afterthe variance between groups is compared by an F-test, with significancedetermined at p≦0.05. The experiment may also be continued past the endof dosing in which case tumor measurements would continue to be recordedto monitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p≦0.05.

3.1.2. Intraperitoneal/Intracranial Tumor Models

Tumor cells are injected intraperitoneally or intracranially on Day 0.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule starting on Day 1. Observations of morbidityand/or mortality are recorded twice daily. Body weights are measured andrecorded twice weekly. Morbidity/mortality data is expressed in terms ofthe median time of survival and the number of long-term survivors isindicated separately. Survival times are used to generate Kaplan-Meiercurves. Significance is p≦0.05 by a log-rank test compared to thecontrol group in the experiment.

3.2. Established Disease Model

Tumor cells or fragments are implanted subcutaneously and grown to thedesired size for treatment to begin. Once at the predetermined sizerange, mice are randomized into treatment groups. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. according to apredetermined schedule. Tumor and body weights are measured and recorded2-3 times weekly. Mean tumor weights of all groups over days postinoculation are graphed for comparison. An F-test is preformed todetermine if the variance is equal or unequal followed by a Student'st-test to compare tumor sizes in the treated and control groups at theend of treatment. Significance is p≦0.05 as compared to the controlgroup. Tumor measurements may be recorded after dosing has stopped tomonitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p value≦0.05 compared to the vehiclecontrol group.

3.3. Orthotopic Disease Models

3.3.1. Mammary Fat Pad Assay

Tumor cells or fragments, of mammary adenocarcinoma origin, areimplanted directly into a surgically exposed and reflected mammary fatpad in rodents. The fat pad is placed back in its original position andthe surgical site is closed. Hormones may also be administered to therodents to support the growth of the tumors. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.Tumor and body weights are measured and recorded 2-3 times weekly. Meantumor weights of all groups over days post inoculation are graphed forcomparison. An F-test is preformed to determine if the variance is equalor unequal followed by a Student's t-test to compare tumor sizes in thetreated and control groups at the end of treatment. Significance isp≦0.05 as compared to the control group.

Tumor measurements may be recorded after dosing has stopped to monitortumor growth delay. Tumor growth delays are expressed as the differencein the median time for the treated and control groups to attain apredetermined size divided by the median time for the control group toattain that size. Growth delays are compared by generating Kaplan-Meiercurves from the times for individual tumors to attain the evaluationsize. Significance is p value≦0.05 compared to the vehicle controlgroup. In addition, this model provides an opportunity to increase therate of spontaneous metastasis of this type of tumor. Metastasis can beassessed at termination of the study by counting the number of visiblefoci per target organ, or measuring the target organ weight. The meansof these endpoints are compared by Student's t-test after conducting anF-test, with significance determined at p≦0.05 compared to the controlgroup in the experiment.

3.3.2. Intraprostatic Assay

Tumor cells or fragments, of prostatic adenocarcinoma origin, areimplanted directly into a surgically exposed dorsal lobe of the prostatein rodents. The prostate is externalized through an abdominal incisionso that the tumor can be implanted specifically in the dorsal lobe whileverifying that the implant does not enter the seminal vesicles. Thesuccessfully inoculated prostate is replaced in the abdomen and theincisions through the abdomen and skin are closed. Hormones may also beadministered to the rodents to support the growth of the tumors.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule. Body weights are measured and recorded 2-3times weekly. At a predetermined time, the experiment is terminated andthe animal is dissected. The size of the primary tumor is measured inthree dimensions using either a caliper or an ocular micrometer attachedto a dissecting scope. An F-test is preformed to determine if thevariance is equal or unequal followed by a Student's t-test to comparetumor sizes in the treated and control groups at the end of treatment.Significance is p≦0.05 as compared to the control group. This modelprovides an opportunity to increase the rate of spontaneous metastasisof this type of tumor. Metastasis can be assessed at termination of thestudy by counting the number of visible foci per target organ (i.e., thelungs), or measuring the target organ weight (i.e., the regional lymphnodes). The means of these endpoints are compared by Student's t-testafter conducting an F-test, with significance determined at p≦0.05compared to the control group in the experiment.

3.3.3. Intrabronchial Assay

Tumor cells of pulmonary origin may be implanted intrabronchially bymaking an incision through the skin and exposing the trachea. Thetrachea is pierced with the beveled end of a 25 gauge needle and thetumor cells are inoculated into the main bronchus using a flat-ended 27gauge needle with a 90° bend. Compounds are administered p.o., i.p.,i.v., i.m., or s.c. according to a predetermined schedule. Body weightsare measured and recorded 2-3 times weekly. At a predetermined time, theexperiment is terminated and the animal is dissected. The size of theprimary tumor is measured in three dimensions using either a caliper oran ocular micrometer attached to a dissecting scope. An F-test ispreformed to determine if the variance is equal or unequal followed by aStudent's t-test to compare tumor sizes in the treated and controlgroups at the end of treatment. Significance is p≦0.05 as compared tothe control group. This model provides an opportunity to increase therate of spontaneous metastasis of this type of tumor. Metastasis can beassessed at termination of the study by counting the number of visiblefoci per target organ (i.e., the contralateral lung), or measuring thetarget organ weight. The means of these endpoints are compared byStudent's t-test after conducting an F-test, with significancedetermined at p≦0.05 compared to the control group in the experiment.

3.3.4. Intracecal Assay

Tumor cells of gastrointestinal origin may be implanted intracecally bymaking an abdominal incision through the skin and externalizing theintestine. Tumor cells are inoculated into the cecal wall withoutpenetrating the lumen of the intestine using a 27 or 30 gauge needle.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule. Body weights are measured and recorded 2-3times weekly. At a predetermined time, the experiment is terminated andthe animal is dissected. The size of the primary tumor is measured inthree dimensions using either a caliper or an ocular micrometer attachedto a dissecting scope. An F-test is preformed to determine if thevariance is equal or unequal followed by a Student's t-test to comparetumor sizes in the treated and control groups at the end of treatment.Significance is p≦0.05 as compared to the control group. This modelprovides an opportunity to increase the rate of spontaneous metastasisof this type of tumor. Metastasis can be assessed at termination of thestudy by counting the number of visible foci per target organ (i.e., theliver), or measuring the target organ weight. The means of theseendpoints are compared by Student's t-test after conducting an F-test,with significance determined at p≦0.05 compared to the control group inthe experiment.

4. Secondary (Metastatic) Antitumor Efficacy

4.1. Spontaneous Metastasis

Tumor cells are inoculated s.c. and the tumors allowed to grow to apredetermined range for spontaneous metastasis studies to the lung orliver. These primary tumors are then excised. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedulewhich may include the period leading up to the excision of the primarytumor to evaluate therapies directed at inhibiting the early stages oftumor metastasis. Observations of morbidity and/or mortality arerecorded daily. Body weights are measured and recorded twice weekly.Potential endpoints include survival time, numbers of visible foci pertarget organ, or target organ weight. When survival time is used as theendpoint the other values are not determined. Survival data is used togenerate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank testcompared to the control group in the experiment. The mean number ofvisible tumor foci, as determined under a dissecting microscope, and themean target organ weights are compared by Student's t-test afterconducting an F-test, with significance determined at p≦0.05 compared tothe control group in the experiment for both of these endpoints.

4.2. Forced Metastasis

Tumor cells are injected into the tail vein, portal vein, or the leftventricle of the heart in experimental (forced) lung, liver, and bonemetastasis studies, respectively. Compounds are administered p.o., i.p.,i.v., i.m., or s.c. according to a predetermined schedule. Observationsof morbidity and/or mortality are recorded daily. Body weights aremeasured and recorded twice weekly. Potential endpoints include survivaltime, numbers of visible foci per target organ, or target organ weight.When survival time is used as the endpoint the other values are notdetermined. Survival data is used to generate Kaplan-Meier curves.Significance is p≦0.05 by a log-rank test compared to the control groupin the experiment. The mean number of visible tumor foci, as determinedunder a dissecting microscope, and the mean target organ weights arecompared by Student's t-test after conducting an F-test, withsignificance at p≦0.05 compared to the vehicle control group in theexperiment for both endpoints.

EXAMPLE 8 Proliferation Inhibition Assay: Antisense OligonucleotidesSuppress the Growth of Cancer Cell Lines

The cell line used for testing is the human colon cancer cell lineHCT116. Cells are cultured in RPMI-1640 with 10-15% fetal calf serum ata concentration of 10,000 cells per milliliter in a volume of 0.5 ml andkept at 37° C. in a 95% air/5% CO₂ atmosphere.

Phosphorothioate oligoribonucleotides are synthesized on an AppliedBiosystems Model 380B DNA synthesizer using phosphoroamidite chemistry.A sequence of 24 bases complementary to the nucleotides at position 1 to24 of SEQ ID NO:1 or SEQ ID NO: 5 is used as the test oligonucleotide.As a control, another (random) sequence is used: 5′-TCA ACT GAC TAG ATGTAC ATG GAC-3′ (SEQ ID NO:7). Following assembly and deprotection,oligonucleotides are ethanol-precipitated twice, dried, and suspended inphosphate buffered saline at the desired concentration. Purity of theoligonucleotides is tested by capillary gel electrophoresis and ionexchange HPLC. The purified oligonucleotides are added to the culturemedium at a concentration of 10 μM once per day for seven days.

The addition of the test oligonucleotide for seven days results insignificantly reduced expression of human prostasin-like serine proteaseas determined by Western blotting. This effect is not observed with thecontrol oligonucleotide. After 3 to 7 days, the number of cells in thecultures is counted using an automatic cell counter. The number of cellsin cultures treated with the test oligonucleotide (expressed as 100%) iscompared with the number of cells in cultures treated with the controloligonucleotide. The number of cells in cultures treated with the testoligonucleotide is not more than 30% of control, indicating that theinhibition of human prostasin-like serine protease has ananti-proliferative effect on cancer cells.

EXAMPLE 9 In Vivo Testing of Compounds/Target Validation

1. Pain:

Acute Pain

Acute pain is measured on a hot plate mainly in rats. Two variants ofhot plate testing are used: In the classical variant animals are put ona hot surface (52 to 56?C) and the latency time is measured until theanimals show nocifensive behavior, such as stepping or foot licking. Theother variant is an increasing temperature hot plate where theexperimental animals are put on a surface of neutral temperature.Subsequently this surface is slowly but constantly heated until theanimals begin to lick a hind paw. The temperature which is reached whenhind paw licking begins is a measure for pain threshold.

Compounds are tested against a vehicle treated control group. Substanceapplication is performed at different time points via differentapplication routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal,transdermal) prior to pain testing.

Persistent Pain

Persistent pain is measured with the formalin or capsaicin test, mainlyin rats. A solution of 1 to 5% formalin or 10 to 100 μg capsaicin isinjected into one hind paw of the experimental animal. After formalin orcapsaicin application the animals show nocifensive reactions likeflinching, licking and biting of the affected paw. The number ofnocifensive reactions within a time frame of up to 90 minutes is ameasure for intensity of pain.

Compounds are tested against a vehicle treated control group. Substanceapplication is performed at different time points via differentapplication routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal,transdermal) prior to formalin or capsaicin administration.

Neuropathic Pain

Neuropathic pain is induced by different variants of unilateral sciaticnerve injury mainly in rats. The operation is performed underanesthesia. The first variant of sciatic nerve injury is produced byplacing loosely constrictive ligatures around the common sciatic nerve.The second variant is the tight ligation of about the half of thediameter of the common sciatic nerve. In the next variant, a group ofmodels is used in which tight ligations or transections are made ofeither the L5 and L6 spinal nerves, or the L % spinal nerve only. Thefourth variant involves an axotomy of two of the three terminal branchesof the sciatic nerve (tibial and common peroneal nerves) leaving theremaining sural nerve intact whereas the last variant comprises theaxotomy of only the tibial branch leaving the sural and common nervesuninjured. Control animals are treated with a sham operation.

Postoperatively, the nerve injured animals develop a chronic mechanicalallodynia, cold allodynia, as well as a thermal hyperalgesia. Mechanicalallodynia is measured by means of a pressure transducer (electronic vonFrey Anesthesiometer, IITC Inc.—Life Science Instruments, WoodlandHills, SA, USA; Electronic von Frey System, Somedic Sales AB, Hörby,Sweden). Thermal hyperalgesia is measured by means of a radiant heatsource (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a coldplate of 5 to 10 ?C where the nocifensive reactions of the affected hindpaw are counted as a measure of pain intensity. A further test for coldinduced pain is the counting of nocifensive reactions, or duration ofnocifensive responses after plantar administration of acetone to theaffected hind limb. Chronic pain in general is assessed by registeringthe circadanian rhythms in activity (Sujo and Arndt, Universität zuKöln, Cologne, Germany), and by scoring differences in gait (foot printpatterns; FOOTPRINTS program, Klapdor et al., 1997. A low cost method toanalyse footprint patterns. J. Neurosci. Methods 75, 49-54).

Compounds are tested against sham operated and vehicle treated controlgroups. Substance application is performed at different time points viadifferent application routes (i.v., i.p., p.o., i.t., i.c.v., s.c.,intradermal, transdermal) prior to pain testing.

Inflammatory Pain

Inflammatory pain is induced mainly in rats by injection of 0.75 mgcarrageenan or complete Freund's adjuvant into one hind paw. The animalsdevelop an edema with mechanical allodynia as well as thermalhyperalgesia Mechanical allodynia is measured by means of a pressuretransducer (electronic von Frey Anesthesiometer, IITC Inc.—Life ScienceInstruments, Woodland Hills, SA, USA). Thermal hyperalgesia is measuredby means of a radiant heat source (Plantar Test, Ugo Basile, Comerio,Italy, Paw thermal stimulator, G. Ozali, University of California, USA).For edema measurement two methods are being used. In the first method,the animals are sacrificed and the affected hindpaws sectioned andweighed. The second method comprises differences in paw volume bymeasuring water displacement in a plethysmometer (Ugo Basile, Comerio,Italy).

Compounds are tested against uninflamed as well as vehicle treatedcontrol groups. Substance application is performed at different timepoints via different application routes (i.v., i.p., p.o., i.t., i.c.v.,s.c., intradermal, transdermal) prior to pain testing.

Diabetic Neuropathic Pain

Rats treated with a single intraperitoneal injection of 50 to 80 mg/kgstreptozotocin develop a profound hyperglycemia and mechanical allodyniawithin 1 to 3 weeks. Mechanical allodynia is measured by means of apressure transducer (electronic von Frey Anesthesiometer, IITCInc.—Life-Science Instruments, Woodland Hills, SA, USA).

Compounds are tested against diabetic and non-diabetic vehicle treatedcontrol groups. Substance application is performed at different timepoints via different application routes (i.v., i.p., p.o., i.t., i.c.v.,s.c., intradermal, transdermal) prior to pain testing.

2. Parkinson's Disease

6-Hydroxydopamine (6-OH-DA) Lesion

Degeneration of the dopaminergic nigrostriatal and striatopallidalpathways is the central pathological event in Parkinson's disease. Thisdisorder has been mimicked experimentally in rats usingsingle/sequential unilateral stereotaxic injections of 6-OH-DA into themedium forebrain bundle (MFB).

Male Wistar rats (Harlan Winkelmann, Germany), weighing 200±250 g at thebeginning of the experiment, are used. The rats are maintained in atemperature- and humidity-controlled environment under a 12 h light/darkcycle with free access to food and water when not in experimentalsessions. The following in vivo protocols are approved by thegovernmental authorities. All efforts are made to minimize animalsuffering, to reduce the number of animals used, and to utilizealternatives to in vivo techniques.

Animals are administered pargyline on the day of surgery (Sigma, St.Louis, Mo., USA; 50 mg/kg i.p.) in order to inhibit metabolism of 6-OHDAby monoamine oxidase and desmethylimipramine HCl (Sigma; 25 mg/kg i.p.)in order to prevent uptake of 6-OHDA by noradrenergic terminals. Thirtyminutes later the rats are anesthetized with sodium pentobarbital (50mg/kg) and placed in a stereotaxic frame. In order to lesion the DAnigrostriatal pathway 4 μl of 0.01% ascorbic acid-saline containing 8 μgof 6-OHDA HBr (Sigma) are injected into the left medial fore-brainbundle at a rate of 1 μl/min (2.4 mm anterior, 1.49 mm lateral, −2.7 mmventral to Bregma and the skull surface). The needle is left in place anadditional 5 min to allow diffusion to occur.

Stepping Test

Forelimb akinesia is assessed three weeks following lesion placementusing a modified stepping test protocol. In brief, the animals are heldby the experimenter with one hand fixing the hindlimbs and slightlyraising the hind part above the surface. One paw is touching the table,and is then moved slowly sideways (5 s for 1 m), first in the forehandand then in the backhand direction. The number of adjusting steps iscounted for both paws in the backhand and forehand direction ofmovement. The sequence of testing is right paw forehand and backhandadjusting stepping, followed by left paw forehand and backhanddirections. The test is repeated three times on three consecutive days,after an initial training period of three days prior to the firsttesting. Forehand adjusted stepping reveals no consistent differencesbetween lesioned and healthy control animals. Analysis is thereforerestricted to backhand adjusted stepping.

Balance Test

Balance adjustments following postural challenge are also measuredduring the stepping test sessions. The rats are held in the sameposition as described in the stepping test and, instead of being movedsideways, tilted by the experimenter towards the side of the pawtouching the table. This maneuver results in loss of balance and theability of the rats to regain balance by forelimb movements is scored ona scale ranging from 0 to 3. Score 0 is given for a normal forelimbplacement. When the forelimb movement is delayed but recovery ofpostural balance detected, score 1 is given. Score 2 represents a clear,yet insufficient, forelimb reaction, as evidenced by muscle contraction,but lack of success in recovering balance, and score 3 is given for noreaction of movement. The test is repeated three times a day on eachside for three consecutive days after an initial training period ofthree days prior to the first testing.

Staircase Test (Paw Reaching)

A modified version of the staircase test is used for evaluation of pawreaching behavior three weeks following primary and secondary lesionplacement. Plexiglass test boxes with a central platform and a removablestaircase on each side are used. The apparatus is designed such thatonly the paw on the same side at each staircase can be used, thusproviding a measure of independent forelimb use. For each test theanimals are left in the test boxes for 15 min. The double staircase isfilled with 7×3 chow pellets (Precision food pellets, formula: P,purified rodent diet, size 45 mg; Sandown Scientific) on each side.After each test the number of pellets eaten (successfully retrievedpellets) and the number of pellets taken (touched but dropped) for eachpaw and the success rate (pellets eaten/pellets taken) are countedseparately. After three days of food deprivation (12 g per animal perday) the animals are tested for 11 days. Full analysis is conducted onlyfor the last five days.

MPTP Treatment

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)causes degeneration of mesencephalic dopaminergic (DAergic) neurons inrodents, non-human primates, and humans and, in so doing, reproducesmany of the symptoms of Parkinson's disease. MPTP leads to a markeddecrease in the levels of dopamine and its metabolites, and in thenumber of dopaminergic terminals in the striatum as well as severe lossof the tyrosine hydroxylase (TH)-immunoreactive cell bodies in thesubstantia nigra, pars compacta.

In order to obtain severe and long-lasting lesions, and to reducemortality, animals receive single injections of MPTP, and are thentested for severity of lesion 7-10 days later. Successive MPTPinjections are administered on days 1, 2 and 3. Animals receiveapplication of 4 mg/kg MPTP hydrochloride (Sigma) in saline once daily.All injections are intraperitoneal (i.p.) and the MPTP stock solution isfrozen between injections. Animals are decapitated on day 11.

Immunohistology

At the completion of behavioral experiments, all animals areanaesthetized with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). Themice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min,followed by 4% paraformaldehyde (Merck) in PBS for 15 min. The brainsare removed and placed in 4% paraformaldehyde for 24 h at 4° C. Fordehydration they are then transferred to a 20% sucrose (Merck) solutionin 0.1 M PBS at 4° C. until they sink. The brains are frozen inmethylbutan at −20° C. for 2 min and stored at −70° C. Using a sledgemicrotome (mod. 3800-Frigocut, Leica), 25 μm sections are taken from thegenu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm)and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored inassorters in 0.25 M Tris buffer (pH 7.4) for immunohistochemistry.

A series of sections is processed for free-floating tyrosine hydroxylase(TH) immunohistochemistry. Following three rinses in 0.1 M PBS,endogenous peroxidase activity is quenched for 10 min in 0.3% H₂O₂±PBS.After rinsing in PBS, sections are preincubated in 10% normal bovineserum (Sigma) for 5 min as blocking agent and transferred to eitherprimary anti-rat TH rabbit antiserum (dilution 1:2000).

Following overnight incubation at room temperature, sections for THimmunoreactivity are rinsed in PBS (2×10 min) and incubated inbiotinylated anti-rabbit immunoglobulin G raised in goat (dilution1:200) (Vector) for 90 min, rinsed repeatedly and transferred toVectastain ABC (Vector) solution for 1 h. 3,3′-Diaminobenzidinetetrahydrochloride (DAB; Sigma) in 0.1 M PBS, supplemented with 0.005%H₂O₂ , serves as chromogen in the subsequent visualization reaction.Sections are mounted on to gelatin-coated slides, left to dry overnight,counter-stained with hematoxylin dehydrated in ascending alcoholconcentrations and cleared in butylacetate. Coverslips are mounted onentellan.

Rotarod Test

We use a modification of the procedure described by Rozas andLabandeira-Garcia (1997), with a CR-1 Rotamex system (ColumbusInstruments, Columbus, Ohio) comprising an IBM-compatible personalcomputer, a CIO-24 data acquisition card, a control unit, and afour-lane rotarod unit. The rotarod unit consists of a rotating spindle(diameter 7.3 cm) and individual compartments for each mouse. The systemsoftware allows preprogramming of session protocols with varyingrotational speeds (0-80 rpm). Infrared beams are used to detect when amouse has fallen onto the base grid beneath the rotarod. The system logsthe fall as the end of the experiment for that mouse, and the total timeon the rotarod, as well as the time of the fall and all the set-upparameters, are recorded. The system also allows a weak current to bepassed through the base grid, to aid training.

3. Dementia

The Object Recognition Task

The object recognition task has been designed to assess the effects ofexperimental manipulations on the cognitive performance of rodents. Arat is placed in an open field, in which two identical objects arepresent. The rats inspects both objects during the first trial of theobject recognition task. In a second trial, after a retention intervalof for example 24 hours, one of the two objects used in the first trial,the ‘familiar’ object, and a novel object are placed in the open field.The inspection time at each of the objects is registered. The basicmeasures in the OR task is the time spent by a rat exploring the twoobject the second trial. Good retention is reflected by higherexploration times towards the novel than the ‘familiar’ object.

Administration of the putative cognition enhancer prior to the firsttrial predominantly allows assessment of the effects on acquisition, andeventually on consolidation processes. Administration of the testingcompound after the first trial allows to assess the effects onconsolidation processes, whereas administration before the second trialallows to measure effects on retrieval processes.

The Passive Avoidance Task

The passive avoidance task assesses memory performance in rats and mice.The inhibitory avoidance apparatus consists of a two-compartment boxwith a light compartment and a dark compartment. The two compartmentsare separated by a guillotine door that can be operated by theexperimenter. A threshold of 2 cm separates the two compartments whenthe guillotine door is raised. When the door is open, the illuminationin the dark compartment is about 2 lux. The light intensity is about 500lux at the center of the floor of the light compartment.

Two habituation sessions, one shock session, and a retention session aregiven, separated by inter-session intervals of 24 hours. In thehabituation sessions and the retention session the rat is allowed toexplore the apparatus for 300 sec. The rat is placed in the lightcompartment, facing the wall opposite to the guillotine door. After anaccommodation period of 15 sec. the guillotine door is opened so thatall parts of the apparatus can be visited freely. Rats normally avoidbrightly lit areas and will enter the dark compartment within a fewseconds.

In the shock session the guillotine door between the compartments islowered as soon as the rat has entered the dark compartment with itsfour paws, and a scrambled 1 mA footshock is administered for 2 sec. Therat is removed from the apparatus and put back into its home cage. Theprocedure during the retention session is identical to that of thehabituation sessions.

The step-through latency, that is the first latency of entering the darkcompartment (in sec.) during the retention session is an index of thememory performance of the animal; the longer the latency to enter thedark compartment, the better the retention is. A testing compound ingiven half an hour before the shock session, together with 1 mg*kg⁻¹scopolamine. Scopolamine impairs the memory performance during theretention session 24 hours later. If the test compound increases theenter latency compared with the scopolamine-treated controls, it islikely to possess cognition enhancing potential.

The Morris Water Escape Task

The Morris water escape task measures spatial orientation learning inrodents. It is a test system that has extensively been used toinvestigate the effects of putative therapeutic on the cognitivefunctions of rats and mice. The performance of an animal is assessed ina circular water tank with an escape platform that is submerged about 1cm below the surface of the water. The escape platform is not visiblefor an animal swimming in the water tank. Abundant extra-maze cues areprovided by the furniture in the room, including desks, computerequipment, a second water tank, the presence of the experimenter, and bya radio on a shelf that is playing softly.

The animals receive four trials during five daily acquisition sessions.A trial is started by placing an animal into the pool, facing the wallof the tank. Each of four starting positions in the quadrants north,east, south, and west is used once in a series of four trials; theirorder is randomized. The escape platform is always in the same position.A trial is terminated as soon as the animal had climbs onto the escapeplatform or when 90 seconds have elapsed, whichever event occurs first.The animal is allowed to stay on the platform for 30 seconds. Then it istaken from the platform and the next trial is started. If an animal didnot find the platform within 90 seconds it is put on the platform by theexperimenter and is allowed to stay there for 30 seconds. After thefourth trial of the fifth daily session, an additional trial is given asa probe trial: the platform is removed, and the time the animal spendsin the four quadrants is measured for 30 or 60 seconds. In the probetrial, all animals start from the same start position, opposite to thequadrant where the escape platform had been positioned duringacquisition.

Four different measures are taken to evaluate the performance of ananimal during acquisition training: escape latency, traveled distance,distance to platform, and swimming speed. The following measures areevaluated for the probe trial: time (s) in quadrants and traveleddistance (cm) in the four quadrants. The probe trial provides additionalinformation about how well an animal learned the position of the escapeplatform. If an animal spends more time and swims a longer distance inthe quadrant where the platform had been positioned during theacquisition sessions than in any other quadrant, one concludes that theplatform position has been learned well.

In order to assess the effects of putative cognition enhancingcompounds, rats or mice with specific brain lesions which impaircognitive functions, or animals treated with compounds such asscopolamine or MK-801, which interfere with normal learning, or agedanimals which suffer from cognitive deficits, are used.

The T-Maze Spontaneous Alternation Task

The T-maze spontaneous alternation task (TeMCAT) assesses the spatialmemory performance in mice. The start arm and the two goal arms of theT-maze are provided with guillotine doors which can be operated manuallyby the experimenter. A mouse is put into the start arm at the beginningof training. The guillotine door is closed. In the first trial, the‘forced trial’, either the left or right goal arm is blocked by loweringthe guillotine door. After the mouse has been released from the startarm, it will negotiate the maze, eventually enter the open goal arm, andreturn to the start position, where it will be confined for 5 seconds,by lowering the guillotine door. Then, the animal can choose freelybetween the left and right goal arm (all guillotine-doors opened) during14 ‘free choice’ trials. As soon a the mouse has entered one goal arm,the other one is closed. The mouse eventually returns to the start armand is free to visit whichever goal arm it wants after having beenconfined to the start arm for 5 seconds. After completion of 14 freechoice trials in one session, the animal is removed from the maze.During training, the animal is never handled.

The percent alternations out of 14 trials is calculated. This percentageand the total time needed to complete the first forced trial and thesubsequent 14 free choice trials (in s) is analyzed. Cognitive deficitsare usually induced by an injection of scopolamine, 30 min before thestart of the training session. Scopolamine reduced the per-centalternations to chance level, or below. A cognition enhancer, which isalways administered before the training session, will at leastpartially, antagonize the scopolamine-induced reduction in thespontaneous alternation rate.

EXAMPLE 10 Tissue-Specific Expression of Prostasin-Like Serine Protease

As a first step to establishing a role for prostasin-like serineprotease in the pathogenesis of COPD, expression profiling of the genewas done using real-time quantitative PCR (TaqMan) with RNA samplesisolated from a wide range of human cells and tissues. Total RNA sampleswere either purchased from commercial suppliers or purified in-house.Two panels of RNAs were used for profiling: a whole body organ panel(Table 1.) and a respiratory specific panel (Table 2).

Real-Time Quantitative PCR.

This technique is a development of the kinetic analysis of PCR firstdescribed by Higuchi et al. (BioTechnology 10, 413-17, 1992;BioTechnology 11, 1026-30, 1993). The principle is that at any givencycle within the exponential phase of PCR, the amount of product isproportional to the initial number of template copies. PCR amplificationis performed in the presence of an oligonucleotide probe (TaqMan probe)that is complementary to the target sequence and labeled with afluorescent reporter dye and a quencher dye. During the extension phaseof PCR, the probe is cleaved by the 5′-3′ endonuclease activity of TaqDNA polymerase, releasing the fluorophore from the effect of thequenching dye (Holland et al., Proc. Natl. Acad. Sci. U.S.A. 88,7276-80, 1991). Because the fluorescence emission increases in directproportion to the amount of the specific amplified product, theexponential growth phase of PCR product can be detected and used todetermine the initial template concentration (Heid et al., Genome Res.6, 986-94, 1996, and Gibson et al., Genome Res. 6, 995-1001, 1996).

RNA Extraction and cDNA Preparation.

Total RNA from each of the ‘in-house’ samples listed in Table 2 wereisolated using Qiagen's (Crawley, West Sussex, UK) RNeasy systemaccording to the manufacturer's protocol. The concentration of purifiedRNA was determined using RiboGreen RNA quantitation kit (MolecularProbes Europe, The Netherlands). RNA concentrations of the samplespurchased from commercial suppliers were also determined usingRiboGreen. For the preparation of cDNA, 1 μg of total RNA was reversetranscribed using 200U of SUPERSCRIPT™ RNaseH⁻ Reverse Transcriptase(Life Technologies, Paisley, UK), 10 mM dithiothreitol, 0.5 mM of eachDNTP, and 5 μM random hexamers (PE Applied Biosystems, Warrington,Cheshire, UK) in a final volume of 20 μl according to the manufacturer'sprotocol.

TaqMan Quantitative Analysis.

Specific primers and probe were designed according to therecommendations of PE Applied Biosystems and are listed below: (SEQ IDNO:8) Forward primer: 5′-AGAGTGGCGACCGCTACAAG-3′ (SEQ ID NO:9) Reverseprimer: 5′-TGGTAGAGGCCGTCGCAG-3′ (SEQ ID NO:10) Probe:5′-(FAM)-CGAGTCCAGCAGCGGCACCCTTACT-3′

where FAM=6-carboxy-fluorescein.

Quantification PCR was performed with 10 ng of reverse transcribed RNAfrom each sample. Each determination was done in duplicate.

The assay reaction mix was as follows: 1× final TaqMan Universal PCRMaster Mix (from 2× stock) (PE Applied Biosystems, CA); 900 mM forwardprimer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25μl.

Each of the following steps were carried out once: pre PCR, 2 minutes at50° C., and 10 minutes at 95° C.

The following steps are carried out 40 times: denaturation, 15 secondsat 95° C., annealing/extension, 1 minute at 60° C.

Real-time quantitative PCR was done using an ABI Prism 7700 SequenceDetector.

The CT value generated for each reaction was used to determine theinitial template concentration (copy number) by interpolation from auniversal standard curve. The level of expression of the target gene ineach sample was calculated relative to the sample with the lowestexpression of the gene.

The expression of prostasin-like enzyme in various human tissues isshown in FIG. 12. The highest levels of mRNA were detected in testis,bone marrow, spleen, and lung. Interestingly, expression of the enzymewas not detected in prostate or liver, which are tissues that exhibithigh expression of prostasin itself (Yu et al., J. Biol. Chem. 270,13483-13489). Moreover, prostasin is not expressed in testis, incontrast to the prostasin-like enzyme.

Expression of prostasin-like enzyme in lung was investigated further byanalyzing the expression of the gene in some of the constituent celltypes of the lung. This revealed consistent and abundant expression ofprostasin-like enzyme in polymorphonuclear leukocytes (FIG. 13).Although there was relatively high expression of the gene in circulatingmonocytes from one donor, expression was not detected in monocytes fromtwo other donors. Expression in trachea and alveolar type II cells wasalso clear.

The function of prostasin is unknown, although its widespread expressionsuggests its involvement in important biological processes. It is likelyto be a membrane bound enzyme, indicated by the presence of a putativeC-terminal membrane anchor, and, as such, may be involved in theprocessing of peptides and proteins at epithelial cell surfaces. Theexpression profile of prostasin-like enzyme is quite different to thatof prostasin, perhaps indicating that these two proteases have differentphysiological functions. The relatively high expression of the enzyme inpolymorphonuclear leukoctyes, one of the key inflammatory cell types ofthe lung, suggests that prostasin-like enzyme may be involved ininflammatory processes, and that dysregulation of the protease couldplay a significant role in the destruction of the lung matrix indiseases such as COPD. Prostasin-like enzyme, therefore, represents apotential therapeutic target for COPD. TABLE 1 Human organ RNA panelused for real-time quantitative PCR. All samples were obtained fromClontech UK Ltd, Basingstoke, UK. Tissue Cat. # Adrenal gland HumanPanel V, K4004-1 Bone marrow Human Panel II, K4001-1 Brain Human PanelI, K4000-1 Colon Human Panel II, K4001-1 Heart Human Panel III, K4002-1Kidney Human Panel I, K4000-1 Liver Human Panel I, K4000-1 Lung HumanPanel I, K4000-1 Mammary gland Human Panel III, K4002-1 Pancreas HumanPanel V, K4004-1 Prostate Human Panel III, K4002-1 Salivary gland HumanPanel V, K4004-1 Skeletal muscle Human Panel III, K4002-1 Smallintestine Human Panel II, K4001-1 Spleen Human Panel II, K4001-1 StomachHuman Panel II, K4001-1 Testis Human Panel III, K4002-1 Thymus HumanPanel II, K4001-1 Thyroid Human Panel V, K4004-1 Uterus Human Panel III,K4002-1

TABLE 2 Human respiratory specific RNA panel used for real-timequantitative PCR. Tissue/cell type Supplier, cat # Lung (fetal) Takara(Japan) Lung Clontech, Human Panel I, K4000-1 Trachea Clontech, HumanPanel I, K4000-1 Cultured human bronchial epithelial cells In-houseCultured airway smooth muscle cells In-house Cultured small airwayepithelial cells In-house Primary cultured alveolar type II cellsIn-house Cultured H441 cells (Clara-like) In-house Freshly isolatedpolymorphonuclear In-house leukocytes (neutrophils) Freshly isolatedmonocytes In-house Cultured monocytes (macrophage-like) In-house

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1. An isolated and purified protein comprising a first polypeptidesegment comprising an amino acid sequence selected from the group ofamino acid sequences shown in SEQ ID NOS:2 and
 6. 2. The protein ofclaim 1 further comprising a second polypeptide segment comprising anamino acid sequence which is not an amino acid sequence selected fromthe group of amino acid sequences shown in SEQ ID NOS:2 and 6, whereinthe second polypeptide segment is joined to the first polypeptidesegment by means of a peptide bond.
 3. An isolated and purified proteincomprising an amino acid sequence which differs from an amino acidsequence selected from the group of amino acid sequences shown in SEQ IDNOS:2 and 6 by between one and ten conservative amino acid substitutionsand which has a serine protease activity.
 4. A pharmaceuticalcomposition comprising: a protein comprising an amino acid sequenceselected from the group of amino acid sequences shown in SEQ ID NOS:2and 6; and a pharmaceutically acceptable carrier.